Localization of synapses in rat cortical cultures

Astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex were compared to determine whether differences in the location of neuronal somas, dendrites, axons, synapses or astrocytes, relative to the bulk culture medium, could help to explain the large difference in neuronal susceptibility to glutamate toxicity between the two culture systems. The cultures were processed for electron microscopy, thin sectioned across their depths, and photomontaged. In astrocyte-rich cultures, most of the dendrites, axons and synapses were sequestered from the medium by a nearly continuous layer of astrocyte cell bodies and processes. In contrast, astrocytes did not cover the synapses or neuronal processes in astrocyte-poor cultures. In neither culture system were neuronal cell somas covered by glia. Since neuronal cell somas are freely exposed to the medium in both culture conditions, it seems unlikely that receptors on the somal membrane mediate the greater susceptibility of neurons in astrocyte-poor cultures to glutamate toxicity. The layer of astrocytes in the astrocyte-rich cultures may provide a physical buffer that could hinder diffusion of substances from the medium to the interstitium of the neuropil. This physical buffer combined with avid glutamate uptake mechanisms might allow astrocytes to maintain a sufficiently low concentration of glutamate in the local extracellular space to protect dendrites and synapses in the astrocyte-rich, but not in the astrocyte-poor cultures, from the excitotoxic effects of glutamate. The results of this study demonstrate that local sequestering of neurites and synapses by a physical buffer of astrocytes may help to explain the relative resistance of neurons cultured with astrocytes to glutamate toxicity. A similar physical sequestering by astrocytes, of sensitive regions of neurons in the brain, may help protect neurons from glutamate toxicity in vivo.