Development of Neurons and Glial Cells in Cerebral Cortex, Cultured in the Presence or Absence of Bioelectric Activity: Morphological Observations.

Chronic blockade of bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but the effects of this treatment on non-neuronal cells have not been investigated. To determine which cell types are affected by chronic suppression of BEA, we investigated their morphological development in primary cultures of rat cerebral cortex, grown with or without the sodium channel blocker tetrodotoxin (TTX). Morphological development was monitored by phase-contrast microscopy and by immunofluorescent staining of markers specific for neurons (NSE, MAP2, B-50, and the 200 kD neurofilament protein), astrocytes (GFAP), oligodendrocytes (galactocerebroside), macrophages (ED-1) and fibroblasts (fibronectin). Neurons in control cultures steadily increased in size and elaborated a dense network of axons and dendrites during the first 3 weeks. Astrocytes proliferated strongly and formed a 'bottom-layer' on which other cells grew. Part of the astrocytes migrated into the peripheral area of the culture, but retracted to the centre after 14 days in vitro (DIV). Oligodendrocytes and macrophages also increased in number, but oligodendrocytes were completely lost by 28 DIV. After 3 weeks, axons that had grown into the periphery of the culture gradually retracted and/or degenerated, following the retracting astrocytes. Some of the neurons died after 21 DIV, but a large part persisted until 42 DIV. Upon TTX treatment from 5/6 DIV, cultures with few macrophages showed an increase in the proportion of necrotic nuclei at 14 and 21 DIV. The retraction of peripherally located fibres was accelerated by 3 - 4 days and their degeneration was augmented. Neuronal density decreased to zero between 21 and 42 DIV. Astrocytes showed a clear decrease in density from 28 DIV. Conversely, the density of macrophages was increased about two-fold from 14 DIV. These results indicate that both neurons and glia are affected by chronic TTX treatment.