Further observations on hippocampal neurons in dispersed cell culture

Abstract

The growth of the processes of dissociated hippocampal neurons from 18‐ to 20‐day‐old rat fetuses has been studied in vitro using a method that permits cells to be maintained at relatively low plating densities for periods up to three weeks (Banker and Cowan, '77). Most of the cells are spherical or ovoid when they first attach to the substratum (either polylysine or collagen) but within 24 to 48 hours they begin to put out processes, and by the end of the first week in culture a significant proportion (∼45%) come to resemble normal pyramidal cells with a more‐or‐less triangular shaped soma, a single dominant dendrite‐like process emerging from the apex of the soma, and several “basal dendrites” arising from the opposite pole of the cell. Comparisons of the lengths of these dendrite‐like processes with those of hippocampal cells in the brains of animals sacrificed on the fourth post‐natal day and impregnated by a variant of the Golgi‐Cox method, indicate that in some cases the rate of process formation in vitro approximates that in vivo and that the general form of the neurons is remarkably like that of immature pyramidal cells. After a week in culture a second type of process can be recognized. These tend to be finer than those we have identified as dendrites; they are relatively uniform in diameter, frequently give off branches at right angles, and in the electron microscope can be seen to form synapses upon the larger processes and cell somata. These axon‐like processes differ from the axons of normal pyramidal cells in two important respects: (i) most commonly they arise from one of the dendritic processes, including, on occasion, the putative “apical” dendrites, and only rarely from the base of the perikaryon; (ii) there may be two or more such processes from a single cell. The thicker, tapering processes can be shown (after incubation in ³H‐uridine) to contain large amounts of RNA; newly‐synthesized RNA does not extend into the finer, axon‐like processes.