Nicotinic cholinergic activity is known to influence synaptic events leading to memory formation in brain regions such as hippocampus. My preliminary results suggest previously unknown, basic mechanisms by which nicotine activity controls neuronal development and synaptic maturation. I propose the following specific aims to identify the mechanisms by which nicotinic stimulation, acting through nicotinic receptor a7- and a4p2-nAChRs, influences synapse formation in the developing hippocampus. 1) Examine the ability of a4p2-nAChRs to drive synapse formation on hippocampal neurons. Use postnatal slices in culture to examine the composition, longevity, and fate of spines induced by a4p2-nAChR activation. Determine whether the effect is direct or depends on either glutamatergic or GABAergic signaling. Identify the responsive cell types and the kinds of pre- and postsynaptic components localized at the spines. Verify the results in vivo by quantifying spine numbers and synaptic contacts in hippocampal slices from wildtype and p2-knockout mice. Use FM4-64 dye uptake and patch-clamp recording to assess the functionality of the induced contacts. 2) Test the ability of a7-nAChRs to enhance synaptic maturation. Test the hypothesis that a7-nAChR activation in postnatal hippocampal slices induces postsynaptic maturation including (a) phosphorylation of both CaMKII and GluR1, and (b) recruitment of AMPA receptors, promoting maturation of functional synapses. Determine the longevity of the changes and whether they depend either on GABAergic or glutamatergic signaling. Compare states of hippocampal innervation in wildtype (WT), a7-, P2-, and a7/p2-double knockout (KO) mice during early postnatal life to identify complementary and/or synergistic effects of the two receptor subtypes predicted from results obtained in slice culture. Results from this study are likely to accentuate the prime vulnerabilities of the developing nervous system to chronic nicotine exposure;more importantly, this study may reveal the reasons for those vulnerabilities and how treatment regimens may be designed to treat maladies associated with chronic exposure. The effects of nicotine on synaptic maturation will also have direct relevance for nicotine addiction because the hippocampus, central in the proposed studies, has recently been implicated in processes that mediate both the acute reinforcing properties of drugs of abuse and in drug relapse. Finally, given that hippocampal synapse maturation may underlie addictive behaviors and relapse, understanding the process of maturation may well be a crucial step in designing cost-effective treatments for both addiction and relapse.
