The proposed research concerns the status of cholinergic synapses in the cerebral cortex during aging and the progression of Alzheimer's disease (AD), studies of normal and altered neurochemical mechanisms of pre- and postsynaptic muscarine receptors, and measurements with agents which may prevent or reverse cholinergic dysfunction in aging and AD. Seven aims are proposed. In aim I, new assays for presynaptic M2 and postsynaptic M1 and nicotine receptors will be used to assess these sited in 7 bilateral cortical regions of normal, aged and AD human brains, for the first time. It is likely that an increasing M1/M2 ratio will correlate closely with increasing neuropathology, and prove to be a useful neurochemical measure of cholinergic denervation. The same brains and regions will be used in aim 2 to compare the laminar cortical patterns of declining M2 receptors, increasing neuritic plaques, and remaining postsynaptic receptors, by silver-staining and new autoradiographic (AR) techniques. These studies test hypotheses that changes in cholinergic synapses may be an invariant feature of AD, and that they correlate spatially and temporally with the appearance of plaques as AD progresses. A library of tissue blocks will be obtained for future work with other kinds of synapses. In aim 3, the efficacy of various M1-agonists will be estimated from their ability to promote a GppNHp-sensitive high affinity state of these receptors, both for aim 7 and for possible AD therapy. Effective M1-agonists have not yet been tried in AD. In aim 4, the conditions necessary for estradiol to upregulate cortical cholinergic nerves will be explored in rats, using sex, time, dose and age as variables. Long term goals include trials of estradiol to prevent changes in cholinergic synapses in aging animals and AD. Aim 5 explores downregulation of nicotine receptors in the rat cortex with respect to nicotine dosage and timing. The intent is to see whether these receptors can be repeatedly activated without causing desensitization. Aim 6 concerns the reason for a major loss of high affinity agonist binding to cortical M2 receptors in old Fisher 344 rats, despite normal M2 levels. Pertussis toxin-catalyzed 32-ADP-ribosylation will be used to assess levels of a guanine nucleotide binding protein (Gi) associated with M2, and attempts will be made to stabilize altered agonist-M2-Gi ternary complexes for further studies. Aim 7 is an attempt to isolate and identify the nucleotide binding proteins associated specifically with rabbit brain M1 and M2, via soluble ternary complexes of each. Long term goals include detailed neurochemical work with the M1 postsynaptic mechanism, which promotes phosphoinositide metabolism and changes in cytosolic calcium, and may control growth.