Developmental and regeneration-associated regulation of the limbic system associated membrane protein in explant cultures of the rat brain

Abstract

In the present study we have examined the topographic and temporal patterns of expression of the limbic system associated membrane protein by light and electron microscopic immunocytochemistry in organotypic cultures of the rat brain. The regional, cellular and subcellular distribution of staining in young cultures was similar to that in the intact brain of corresponding age. Since the tissue in vitro is isolated both from afferents and targets, short-term protein expression appears to be regulated by factors intrinsic to the neuron. In culture, the protein was present on the surface of neurons which are physiologically interconnected, such as neurons belonging to the septohippocampal system (cholinergic neurons in the septum and pyramidal and granule cells in the hippocampus). It was also present on the surface of axons and growth cones during process outgrowth. Thus, the limbic system associated membrane protein is expressed in an appropriate spatial and temporal pattern for mediating interactions between growing axons and their targets. The expression of the protein in culture showed some important differences as compared to the intact brain. With increasing age, there was an increasing scattering and disappearance of immunoreactivity in cultures fixed with paraformaldehyde/glutaraldehyde. The decreased immunoreactivity in aged cultures does not appear to reflect decreased protein synthesis, because unfixed and acetone-fixed expiants continued to show immunostaining. Furthermore, dot-blot assays showed similar amounts of immunoreactivity in culture as in the intact brain of corresponding age. Thus, the age-dependent decrease of immunoreactivity may reflect altered insertion of the protein into the membrane or a modification of the epitope recognized by the antibody. There was a rapid increase (within 1 hour) of immunostaining on the surface of sprouting processes following mechanical lesion of mature, unstained axons. The altered distribution after tissue injury could be a means of ensuring specificity of connectivity during nerve fiber regeneration. On the basis of the reported findings, we suggest that system-specific membrane proteins, including the limbic system associated membrane protein, may mediate the formation of specific connections in the brain. Furthermore, we suggest that the reinnervation processes taking place after central nervous system injury may exhibit a similar molecular basis to the development of neural pathways.