Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation

Abstract

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (×9) and volume (×8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 μm 2 and 445 μm 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.