The proposed studies will investigate basic mechanisms of chemical communication and information storage in the mammalian brain. In particular, the studies will address the role of dendritic calcium (Ca) ion influx in responses to neurotransmitters and possible Ca roles in brain development and memory. The studies will be carried out on hippocampal CA1 pyramidal cells from neonatal rats in primary cell culture; Ca influx in response to electrical, chemical and synaptic inputs will be studied by digital video microscopy using the fluorescent Ca indicator fura2. Ca influx will be in relation to features of dendritic structure discernable in the living cells using digital video Nomarski light microscopy. Electron microscopy will be used to investigate structural details at higher resolution in fixed or rapidly frozen cells. Specific chemical probes such as toxins and antibodies with light and electron microscopic labels will be used to study the localization of specific membrane and cytoskeletal molecules. New and unexpected observations on Ca entry in response to neurotransmitter application suggest that significant amounts of Ca may enter dendrites directly through transmitter-operated channels. This contrasts with an earlier expectation that the major Ca influx on dendrites would be due to voltage-dependent Ca channels. One channel which has been found to permit Ca entry is a channel that responds to glutamate (probably the natural transmitter) and N-methyl D-aspartate (NMDA). This channel (called the NMDA channel) is widespread in mammalian neurons and is present on the rat hippocampal CA1 neurons to be studied here. It has been suggested that Ca entry through NMDA channels may mediate effects on ion channels and/or dendritic structure that could underlie long-term potentiation (LTP), a type of synaptic plasticity that is considered an important model for information storage mechanisms in the CNS. The experiments proposed here will test this hypothesis. Three groups of questions will be pursued. (1) How does Ca influx through NMDA channels compare in magnitude and localization to Ca influx through voltage-dependent Ca channels? (2) Can homologs of dendritic spine structure be identified on cultured neurons? If so, are dendritic spines a site of Ca influx? (3) Does Ca influx produce structural changes in dendritic spines? If so, what specific cytoskeletal molecules are involved?
