The goal of this project is to understand the mechanisms by which lead (Pb2+) exerts neurotoxic effects in the central nervous system (CNS) particularly in the developing brain. N-methyl-D-aspartate (NMDA) receptor-ion channels (nAChRs) are very sensitive to inhibition by Pb2+. Whereas the inhibitory effect of Pb2+ on NMDA receptors is apparently due to its action on a Ca2+ site on the receptor, the mechanisms by which Pb2+ inhibits the activation of the alpha7-bearing nAChRs is still unknown and will be investigated in this proposal. Because NMDA receptors and alpha7-bearing nAChR are involved in memory and learning as well as in other forms of neuronal plasticity and development, it is very likely that inhibition of these receptors by Pb2+, particularly during early stages of neuronal maturation, could be associated with the severe learning disabilities caused by this heavy metal. Considering that distal dendritic development of the hippocampal neurons is particularly sensitive to the toxic effects of Pb2+, and that dendritic development(with formation and maturation of dendritic spines) is associated with learning and memory, the following question is raised: Is the expression of Pb2+-sensitive NMDA receptors and nAChRs restricted during development to particular neuronal regions, such as apical and basal dendrites, in distinct hippocampal areas (CA1, CA3, or dentate gyrus)? To answer this questions we will use state-of-the-art infrared microscopy (which allows visualization of axons, dendrites, and dendritic spines) combined with a computer-driven, robotic system of micromanipulators (which enables us to control the positions of patch electrodes and drug-delivery systems at precisely defined regions on the neuronal surfaces). Recordings can then be made of whole-cell and single-channel currents from neurons either visualized in or acutely dissociated from hippocampi of rats at different ages, and we will be able to map the distribution of Pb2+-sensitive nicotinic and NMDA receptors on cell bodies, dendrites, and dendritic spines of hippocampal neurons. Our preliminary studies also indicate that Pb2+ substantially increases spontaneous transmitter release from hippocampal neurons. Taking into account that NMDA receptors and alpha-BGT-sensitive nAChRs present on presynaptic terminals of CNS neurons can modulate the release of a number of neurotransmitters, it is critical to determine whether Pb2+ alters transmitter release by acting at these presynaptic receptors. Therefore, the effects of Pb2+ on spontaneous and evoked transmitter release will be investigated at the level of single synapses on hippocampal neurons, which will be acutely dissociated at various stages of development. We have developed a technique by which neurons can be acutely dissociated by mechanical means (without enzyme treatment) from hippocampi of rats at various ages. These neurons bear many synaptic terminals that are functional and can be electrically stimulated. Altogether these studies should provide the foundation for an understanding of the net effects of Pb2+ on receptor function, synaptic activation and maturation in the developing CNS.
