J. Neurochem. (2011) 117, 1020–1032.
In this paper, we report the characterization of ‘Hi-Spot’ cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that ‘Hi-Spots’ are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.