Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain

Abstract

Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures. Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes. Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15–17, were seeded on to the slice cultures. Combined cultures were maintained for two to 14 days in vitro. Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate–putamen. Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers. Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used. Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate–putamen and less apparent specificity of regional and cortical laminar attachment. Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon. By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex. These studies revealed several different types of cell–tissue interaction. The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue. This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.