Cerebral cortex samples from patients with Alzheimer's disease and from rats after experimental cholinergic denervation of the cerebral cortex exhibited reductions in the presynaptic marker choline acetyltransferase activity and in the number of M2 muscarine receptors, with no change in the number of M1 receptors. These results are in keeping with evidence that M2 receptors function in cholinergic nerve terminals to regulate the release of acetylcholine, whereas M1 receptors are located on postsynaptic cells and facilitate cellular excitation. New M1-selective agonists and M2-selective antagonists directed at post- or presynaptic sites deserve consideration as potential agents for the treatment of the disease.

Evidence suggests that cholinergic input to the hippocampus plays an important role in learning and memory and that degeneration of cholinergic terminals in the hippocampus may contribute to the memory loss associated with Alzheimer's disease. One of the more prominent effects of cholinergic agonists on hippocampal physiology is the potentiation of N-methyl-D-aspartate (NMDA)-receptor currents by muscarinic agonists. Here, we employ traditional pharmacological reagents as well as m1-toxin, an m1 antagonist with unprecedented selectivity, to demonstrate that this potentiation of NMDA-receptor currents in hippocampal CA1 pyramidal cells is mediated by the genetically defined m1 muscarinic receptor. Furthermore, we demonstrate the colocalization of the m1 muscarinic receptor and the NR1a NMDA receptor subunit at the electron microscopic level, indicating a spatial relationship that would allow for physiological interactions between these two receptors. This work demonstrates that the m1-muscarinic receptor gene product modulates excitatory synaptic transmission, and it has important implications in the study of learning and memory as well as the design of drugs to treat neurodegenerative diseases such as Alzheimer's.One of the major neuromodulatory inputs to the hippocampus is a large cholinergic projection from the medial septum and the diagonal band of Broca (1). Both animal and human studies indicate that cholinergic modulation of hippocampal and cortical function plays an important role in memory and attention (2-7). Furthermore, abundant evidence suggests that the clinical syndrome associated with Alzheimer's disease results, at least in part, from the degeneration of basal forebrain cholinergic neurons and the resulting depletion of cholinergic markers in neocortex and hippocampus (8-12). Because of this, a great deal of effort has been focused on determining the cellular mechanisms involved in cholinergic modulation of hippocampal function and the specific acetylcholine (ACh) receptor subtypes that mediate these responses.One of the predominant effects of cholinergic agonists on hippocampal CA1 neurons is potentiation of currents through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor (NMDAR) (13-16). The NMDAR plays a pivotal role in long-lasting forms of synaptic plasticity thought to underlie learning and memory (17). Thus, potentiation of NMDAR currents (I NMDA ) could provide a crucial mechanism by which cholinergic input to the hippocampus modulates memory and attention. In addition, the cholinergic receptor that mediates this potentiation could provide a target for the development of drugs to treat memory disorders (e.g., Alzheimer's disease).Evidence suggests that ACh-induced potentiation of NMDAR currents is mediated by muscarinic ACh receptors (mAChRs) (14). However, the specific mAChR subtype that mediates this response is not known. The mAChRs have been classified into m1-m5 subtypes based on molecular analysis of genes that encode five highly related but structurally distin...

SUMMARY1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [14C]-choline (30 4uM) at 370 0. The synthesis, storage and subsequent release of [I4C]acetylcholine by the muscles was assessed by isotopic-and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0*92,u-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation.[14C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35 % of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm2 sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [14C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0-013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0 43 % of stores/ sec, and amounted to 3 5-7 x 1081 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released. 6. Preformed [3H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.

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