The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

Eyeblink classical conditioning is a relatively simple form of associative learning that has become an invaluable tool in our understanding of the neural mechanisms of learning. When studying rabbits in this paradigm, we observed a dramatic modification of learning rate by conducting training during episodes of either hippocampal theta or hippocampal non-theta activity as determined by on-line slow-wave spectral analysis. Specifically, if animals were given trials only when a computer analysis verified a predominance of slow-wave oscillations at theta frequencies (3-8 Hz), they learned in half as many trials as animals trained during non-theta hippocampal activity (58 vs. 115). This finding provides important evidence from awake, behaving animals that supports recent advances in our knowledge of (i) brain sites and neurobiological mechanisms of learning and memory, specifically hippocampus and theta oscillations, (ii) the biological plausibility of current models of hippocampal function that posit important roles for oscillatory potentials, and (iii) the design of interfaces between biological and cybernetic (electronic) systems that can optimize cognitive processes and performance.

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Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid ␤ (A␤) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which A␤ oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease. Alzheimer disease (AD)2 likely begins with deficits in synaptic transmission in brain regions that are critical for higher cognitive function (1), as stereological analyses of post-mortem samples show that synaptic loss correlates with cognitive dysfunction better than amyloid plaque or neurofibrillary tangle load (2-5). One of the triggers for such synaptic impairments is thought to be soluble oligomers of amyloid ␤ (A␤) peptides that accumulate in the brain and are potently toxic to synapses (6 -8). A␤ synaptotoxicity is consistently observed with A␤ oligomers from synthetic preparations (9 -11), cell-derived forms (7,8,12), and the brains of AD patients (13) and APPswe transgenic mice (14), such that these oligomers disrupt synaptic structure (9, 10, 15-18), inhibit long term potentiation (LTP) (7,8,19), and induce memory deficits (13,20).Although the mechanisms by which A␤ oligomers disrupt synaptic function are not known, A␤ oligomers bind to the dendritic processes of specific subtypes of hippocampal neurons with high affinity (9,15,21), leading to the hypothesi...

Conditioning-specific reflex modification (CRM) occurs when classical conditioning modifies responding to an unconditioned stimulus in the absence of a conditioned stimulus. This form of reflex modification suggests that learning modifies the unconditioned reflex pathway. Rabbit (Oryctolagus cuniculus) nictitating membrane responses to 5 intensities and 3 durations of airpuff (AP) or periorbital electrical stimulation (ES) were monitored before and after conditioning. AP tests detected strong CRM after conditioning with ES and modest levels of CRM after conditioning with AP. After conditioning with AP, ES tests failed to detect CRM. After conditioning with a stronger AP, CRM was again detected by AP tests. CRM is a general phenomenon but is more readily detected after training with a relatively aversive stimulus; thus, it may be a function of level of arousal.

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Conditioning-specific reflex modification (CRM) of the rabbit's nictitating membrane response (NMR) describes changes in responding to an unconditioned stimulus (US) when the rabbit is tested in the absence of the conditioned stimulus. Specifically, after at least 3 days of tone-electrical stimulation pairings, responses to the US increase in size, especially at intensities weaker than the training intensity. CRM is similar to classical conditioning in that it is a function of the strength of conditioning, it can be extinguished, and it can be generalized from one stimulus to another. To compare CRM and classical conditioning further, we conducted three experiments to examine the effects of US intensity (1.0, 2.0, and 4.0 mA) on CRM. CRM was weak following conditioning with 1.0 mA and extremely strong following conditioning with 2.0 mA and 4.0 mA. The data suggest that CRM is a function of US intensity and have implications for posttraumatic stress disorder, a disorder potentially modeled by CRM.

One approach to ameliorate the cognitive decline in Alzheimer's disease (AD) has been to restore neuronal signaling from the basal forebrain cholinergic system via the activation of the M(1) muscarinic receptor. A number of nonselective M(1) muscarinic agonists have previously shown positive effects on cognitive behaviors in AD patients, but were limited due to cholinergic adverse events thought to be mediated by the activation of the M(2) to M(5) subtypes. One strategy to confer selectivity for M(1) is the identification of positive allosteric modulators, which would target an allosteric site on the M(1) receptor rather than the highly conserved orthosteric acetylcholine binding site. Quinoline carboxylic acids have been previously identified as highly selective M(1) positive allosteric modulators with good pharmacokinetic and in vivo properties. Herein is described the optimization of a novel quinolizidinone carboxylic acid scaffold with 4-cyanopiperidines being a key discovery in terms of enhanced activity. In particular, modulator 4i gave high plasma free fractions, enhanced central nervous system (CNS) exposure, was efficacious in a rodent in vivo model of cognition, and afforded good physicochemical properties suitable for further preclinical evaluation.