COMPARTMENTATION OF ALPHA-INTERNEXIN AND NEUROFILAMENT TRIPLET PROTEINS IN CULTURED HIPPOCAMPAL-NEURONS

Intermediate filaments comprise an integral part of the neuronal cytos keleton. However, little is known about their function, and there rema ins some uncertainty about their precise subcellular localization. We examined the timing of expression and distribution of alpha-internexin , neurofilament triplet proteins and peripherin using immunocytochemis try in cultured hippocampal neurons. alpha-Internexin immunostaining w as present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites w hich extended into dendritic spines. The presence of alpha-internexin in dendritic spines was confirmed in situ by electron microscopy of ra t hippocampal tissue sections and suggests that this intermediate fila ment may serve as a link between cytoskeletal elements in dendritic sh afts and spines. In culture, immunostaining using antibodies against i ndividual triplet protein subunits indicated that light (NF-L) and mid dle (NF-M) subunits were first expressed in cells shortly after the in itiation of axonal outgrowth. Expression of the heavy (NF-H) subunit o ccurred a few days later. Although timing and localization of expressi on did not correlate with the initiation of axonal or dendritic proces ses, it was coincident with periods of rapid outgrowth. Triplet protei ns were more abundant in axons and appeared to be incorporated into le ngthier filaments than in dendrites. Highly phosphorylated NFH/M immun oreactivity was polarized to axons after 6 days in culture. The distri bution of one NF-H epitope was restricted to GABAergic neurons in matu re cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results sh ow that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of alpha-internexin suggests that it may be involved in the formation or maintenance of dendritic spines.