Cells dissociated from hippocampal formations of newborn rats are grown in mass cultures (several thousand neurons in a well, 9 mm in diameter) or in microcultures (1 to ca. 20 neurons on islands, 30-300 mm in diameter). Neurons in mass cultures exposed chronically to synaptic blocking agents (kynurenate and elevated Mg(2+)) exhibit intense seizure-like activity when the agents are withdrawn (after several weeks or months). The activity has two major components: paroxysmal depolarization shifts (PDSS) occurring synchronously in most or all neurons of the population; sustained depolarizations (10-60 mV) occurring in many neurons. If this activity is allowed to persist too long, it kills most of the neurons. We are interested in the factors that make the cultures seizure-prone, and that cause the death of the neurons. Is the presence of the activity in the blocked cultures due to selective survival of certain types of neurons or to blocker-induced changes in the properties of the surviving neurons? To address this question we will examine the proportions of excitatory and inhibitory neurons in blocked and unblocked cultures, and examine the anatomical and physiological properties of individual neurons. We will determine the minimal exposure period for the blockers to induce the seizure-like activity, examine whether there is a critical period when the exposure is effective, and test whether other agents (e.g. anticonvulsants, tetrodotoxin, other glutamate-receptor antagonists) are effective. During the seizure-associated sustained depolarizations there appears to be a continued release of glutamate (or related agonist), often in the absence of Na+-dependent action potentials. What is the source of this agonist, and does its continued release contribute to the death of the neurons? We will use Ca2+-sensitive fluorescent dyes to explore the relationship between the seizure-like activity and the accumulation of intracellular Ca2+, and between Ca2+ accumulation and subsequent neuronal death. Observations on microcultures have shown that only a few neurons, and in some cases only a single neuron, are needed to generate major aspects of the seizure-like activity. Microcultures will be used to examine in greater detail the cellular mechanisms that underlie the seizure-like activity, with emphasis on comparing the complements of neurotransmitter receptors in blocked and unblocked neurons. We will also explore whether excitatory or inhibitory neurons, or neurons from different regions of the hippocampal formation, show consistent differences in their types of transmitter receptors when cultured under the same conditions.
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