Meeting Report for: The 13th International Symposium on Cholinergic Mechanisms

Neurotoxicity induced by glutamate and other excitatory amino acids has been implicated in various neurodegenerative disorders including hypoxic ischemic events, trauma, and Alzheimer's and Parkinson's diseases. We examined the roles of nicotinic acetylcholine receptors (nAChRs) in survival of CNS neurons during excitotoxic events. Nicotine as well as other nicotinic receptor agonists protected cortical neurons against glutamate neurotoxicity via alpha4 and alpha7 nAChRs at least partly by inhibiting the process of apoptosis in near-pure neuronal cultures obtained from the cerebral cortex of fetal rats. Donepezil, galanatamine and tacrine, therapeutic acetylcholinesterase (AChE) inhibitors currently being used for treatment of Alzheimer's disease also protected neuronal cells from glutamate neurotoxicity. Protective effects of nicotine and the AChE inhibitors were antagonized by nAChR antagonists. Moreover, nicotine and those AChE inhibitors induced up-regulation of nAChRs. Inhibitors for a non-receptor-type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galantamine. Furthermore, a phosphatidylinositol 3-kinase (PI3K) inhibitor also suppressed the neuroprotective effect of the AChE inhibitors. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galantamine treatments. These results suggest that nicotine as well as AChE inhibitors, donepezil and galantamine, prevent glutamate neurotoxicity through alpha4 and alpha7 nAChRs and the PI3K-Akt pathway.

It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different posttranslational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal "alpha(4)beta(2)" and "alpha(7)" subtypes most abundant in brain. The loss of ligand binding sites that occurs when palmitoylation is blocked with the inhibitor bromopalmitate suggests that palmitoylation of alpha(4)beta(2) and alpha(7) subtypes occurs during subunit assembly and regulates the formation of ligand binding sites. However, additional experiments are needed to test whether nAChR subunit palmitoylation is involved in other aspects of nAChR trafficking or whether palmitoylation regulates nAChR function. Further investigation would be aided by identifying the sites of palmitoylation on the subunits, and here we propose a mass spectrometry strategy for identification of these sites.

In mammals, at least five different muscarinic acetylcholine receptor subtypes (mAChRs; M(1)-M(5)) are known to be widely expressed and distributed in different tissues from different species. They mediate distinct physiological functions according to their location and receptor subtype. Multiple events are associated with the regulation of intracellular signaling by mAChRs, and a coordinated balance of the molecular mechanisms governing receptor signaling, desensitization, resensitization, and mitogenic signaling is known to occur in various cell types. Most of the actions of acetylcholine (ACh) in the male reproductive tract are induced by its effects on mAChRs, but the role of specific mAChR subtypes on male reproductive function and fertility are still not well understood. The rat efferent ductules and epididymis are androgen-dependent tissues of the male reproductive tract, with important roles in the process to form a viable and fertile sperm. In the present study, aspects of the expression, localization, and potential function of mAChR subtypes in rat efferent ductules and epididymis are reviewed. Furthermore, evidences for the implication of mAChRs in the regulation of protein synthesis and secretion in these tissues are presented. Taken together, the studies contribute to our understanding about physiological aspects of mAChR and mechanisms by which the cholinergic system affects male reproduction.

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal ("shell" or "annular") lipids, which occur in the liquid-ordered phase as opposed to the more disordered and "fluid" bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.

Basal forebrain cholinergic neurons are highly dependent on nerve growth factor (NGF) supply for the maintenance of their cholinergic phenotype as well as their cholinergic synaptic integrity. The precursor form of NGF, proNGF, abounds in the CNS and is highly elevated in Alzheimer's disease. In order to obtain a deeper understanding of the NGF biology in the CNS, we have performed a series of ex vivo and in vivo investigations to elucidate the mechanisms of release, maturation and degradation of this neurotrophin. In this short review, we describe this NGF metabolic pathway, its significance for the maintenance of basal cholinergic neurons, and its dysregulation in Alzheimer's disease. We are proposing that the conversion of proNGF to mature NGF occurs in the extracellular space by the coordinated action of zymogens, convertases, and endogenous regulators, which are released in the extracellular space in an activity-dependent fashion. We further discuss our findings of a diminished conversion of the NGF precursor molecule to its mature form in Alzheimer's disease as well as an augmented degradation of mature NGF. These combined effects on NGF metabolism would explain the well-known cholinergic atrophy found in Alzheimer's disease and would offer new therapeutic opportunities aimed at correcting the NGF dysmetabolism along with Abeta-induced inflammatory responses.

Mesolimbic dopamine (DA) systems play a critical role in tobacco addiction driven by nicotine. Nicotine activates midbrain DA neurons and, consequently, elevates DA concentrations in targets, especially in the nucleus accumbens (NAc) of the ventral striatum. The route of drug administration influences the impact of addictive drugs. Here, we examine whether the nature of the administration alters DA neuron activity and DA concentrations in the NAc. Using unhabituated naive freely moving rats, microdialysis measurements showed that nicotine administered via needle injection caused greater DA release in the NAc than the same dose administered via an implanted chronic cannula. After habituation to the needle injections, however, there was no significant difference in DA signaling between the needle and cannula routes of administration. Consistent with these microdialysis results after habituation, our in vivo tetrode unit recordings showed no significant difference in midbrain DA neuron activity in response to nicotine delivered by needle or cannula as long as predictive cues were avoided

Although synaptic transmission was assumed to use the same mechanisms in the case of different synapses of the central and peripheral nervous system, recent research revealed a great variety of different processes. Time might be a crucial factor to be considered in this diversity. It is recalled that the speed of a chemical reaction is inversely related to affinity. "Time is gained at the expense of sensitivity" as noticed by Bernard Katz (1989). Therefore, synaptic transmission will occur at a high speed only if it is supported by low affinity reactions. In the present work, we compare two examples of ultra-rapid transmission (the Torpedo nerve electroplaque synapse and the rat hippocampus mossy fiber/CA3 synapses), with a cholinergic process operating with high affinity but at a low speed: the release of glutamate elicited by nicotine from mossy fibers of the rat hippocampus.

The congenital myasthenic syndromes have now been traced to an array of molecular targets at the neuromuscular junction encoded by no fewer than 11 disease genes. The disease genes were identified by the candidate gene approach, using clues derived from clinical, electrophysiological, cytochemical, and ultrastructural features. For example, electrophysiologic studies in patients suffering from sudden episodes of apnea pointed to a defect in acetylcholine resynthesis and CHAT as the candidate gene (Ohno et al., Proc Natl Acad Sci USA 98:2017-2022, 2001); refractoriness to anticholinesterase medications and partial or complete absence of acetylcholinesterase (AChE) from the endplates (EPs) has pointed to one of the two genes (COLQ and ACHE ( T )) encoding AChE, though mutations were observed only in COLQ. After a series of patients carrying mutations in a disease gene have been identified, the emerging genotype-phenotype correlations provided clues for targeted mutation analysis in other patients. Mutations in EP-specific proteins also prompted expression studies that proved pathogenicity, highlighted important functional domains of the abnormal proteins, and pointed to rational therapy.

Wild-type and alpha5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate alpha-conotoxin MII (alpha-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration-effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the alpha5 null mutant. Deleting the alpha5 gene also resulted in decreased maximal ACh-stimulated alpha-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and alpha5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHbetaE-sensitive (HS) and DHbetaE-resistant (LS) components were found in both genotypes. These results indicate that alpha-CtxMII-resistant DA release is mediated by alpha4alpha5beta2, (alpha4)(2)(beta2)(3) (HS), and (alpha4)(3)(beta2)(2) (LS) nAChRs.

Nicotinic acetylcholine receptor (nAChR) modulation of a number of parameters of synaptic signaling in the brain has been demonstrated. It is likely that effects of nicotine are due to its ability to modulate network excitability as a whole. A pre-requisite to understanding the effects of nicotine on network properties is the elucidation of functional receptors. We have examined the distribution of functional nAChRs in the dentate gyrus granule cells and the CA3 region of the mammalian hippocampus using calcium imaging from acute slices. Our results demonstrate the presence of functional nAChRs containing the alpha7 subunit (alpha7-nAChRs) on mossy fiber boutons, CA3 pyramidal cells, and on astrocytes. In addition, both CA3 interneurons and granule cells show nicotinic signals. Our study suggests that functional nicotinic receptors are widespread in their distribution and that calcium imaging might be an effective technique to examine locations of these receptors in the mammalian brain.

Dysfunctional cholinergic transmission is thought to underlie, at least in part, memory impairment and cognitive deficits in Alzheimer's disease (AD). However, it is still unclear whether this is a consequence of the loss of cholinergic neurons and elimination of nicotinic acetycholine receptors (nAChRs) in AD brain or of a direct impact of molecular interactions of the amyloid-beta (Abeta) peptide with nAChRs, leading to dysregulation of receptor function. This review examines recent progress in our understanding of the roles of nicotinic receptors in mechanisms of synaptic plasticity, molecular interactions of Abeta with nAChRs, and how Abeta-induced dysregulation of nicotinic receptor function may underlie synaptic failure in AD.

In the course of the five most recent International Symposia on Cholinergic Mechanisms (ISCMs) as well as at this XIII ISCM, the focus was mostly on matters microscopic and molecular, except for solitary excursions into memory and learning, addiction, and clinical and toxicological subjects. In fact, this is also true for many recent reviews and meetings concerned with the cholinergic transmission. Yet, macroscopic and overt phenomena that are of great importance have strong cholinergic correlates. To remedy this lacuna, macromolecular overt cholinergic brain-dependent activities will be reviewed such as reflex automatic phenomena, EEG functions, behaviors, and the sense of the "self" or "self-awareness", particularly the difficulties associated with neuroscientific explanation of the "self," and the many approaches to this dilemma will be discussed.

The mammalian muscle nicotinic acetylcholine receptor (AChR) is composed of five membrane-spanning subunits and its composition differs between embryonic and adult muscles. In embryonic muscles, it is composed of two alpha-, one beta-, one delta-, and one gamma-subunit; the gamma-subunit is later replaced by the epsilon-subunit during postnatal development. This unique temporal expression pattern of the gamma-subunit suggests it may play specific roles in embryonic muscles. To address this issue, we examined the formation and function of the neuromuscular junction in mouse embryos deficient in the gamma-subunit. At embryonic day 15.5, AChR clusters were absent and the spontaneous miniature endplate potentials were undetectable in the mutant muscles. However, electrical stimulation of the nerves triggered muscle contraction and elicited postsynaptic endplate potential (EPP) in the mutant muscles, although the magnitude of the muscle contraction and the amplitudes of the EPPs were smaller in the mutant compared to the wild-type muscles. Reintroducing a wild-type gamma-subunit into the mutant myotubes restored the formation of AChR clusters in vitro. Together, these results have demonstrated that functional AChRs were present in the mutant muscle membrane, but at reduced levels. Thus, in the absence of the gamma-subunit, a combination of alpha, beta, and delta subunits may assemble into functional receptors in vivo. These results also suggest that the gamma-subunit maybe involved in interacting with rapsyn, a cytoplasmic protein required for AChR clustering.

The aim of this study was to explore the modulation by alpha7 nicotinic receptors (nAChRs) of dopamine and glutamate release in the rat prefrontal cortex where these receptors are implicated in attentional processes and are therapeutic targets for cognitive deficits. The presence of presynaptic alpha7 nAChRs on glutamate terminals is supported by the ability of the subtype-selective agonist Compound A to evoke [(3)H]D-aspartate release from synaptosomes: This response was potentiated by the selective allosteric potentiator PNU-120596 and blocked by alphabungarotoxin. Compound A also evoked dopamine overflow in the prefrontal cortex in vivo, and this was potentiated by PNU-120596. alpha7 nAChR-evoked [(3)H]dopamine release from tissue prisms in vitro was blocked by antagonists of NMDA and AMPA receptors. These data are consistent with a model in which alpha7 nAChRs present on glutamate terminals increase glutamate release that (1) contributes to presynaptic facilitation and synaptic plasticity and (2) co-ordinately enhances dopamine release from neighbouring boutons.

The main immunogenic region on the alpha1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation dependence of its epitopes by making chimeras in which sequences of human muscle alpha1 subunits were replaced in human neuronal alpha7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation and, consequently, assembly of receptor subunits.

Memogain (Gln-1062) is an inactive pro-drug of galantamine, the latter being a plant alkaloid approved for the treatment of mild to moderate Alzheimer's disease. Memogain has more than 15-fold higher bioavailability in the brain than the same doses of galantamine. In the brain, Memogain is enzymatically cleaved to galantamine, thereby regaining its pharmacological activity as a cholinergic enhancer. In animal models of drug-induced amnesia, Memogain produced several fold larger cognitive improvement than the same doses of galantamine, without exhibiting any significant levels of gastrointestinal side effects that are typical for the unmodified drug and other inhibitors of cholinesterases, such as donepezil and rivastigmin. In the ferret, dramatically reduced emetic and behavioral responses were observed when Memogain was administered instead of galantamine. Based on these and other preclinical data, Memogain may represent an advantageous drug treatment for Alzheimer's disease, combining much lesser gastrointestinal side effects and considerably higher potency in enhancing cognition, as compared to presently available drugs.

Use of tobacco products contributes to hundreds of thousands of premature deaths and untold millions of dollars in health care costs in this country each year. Nicotine is the principal neuroactive component in tobacco, but, despite ongoing research efforts, the cellular basis of its effects on behavior remains unclear. Efforts to resolve this conundrum have focused on the mesoaccumbens dopamine system, which contributes to the rewarding effects of many addictive drugs, including nicotine. The goal of this review is to outline recent advances and highlight some of the important unanswered questions regarding nicotine's effects on neuronal excitability and synaptic plasticity within the brain reward pathways.

The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the alpha7, beta2, or beta4 subunit. In agreement with general belief, the alpha4beta2*-nAChR and alpha7-nAChR subtypes are major components of the epibatidine binding sites. alpha4beta2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while alpha7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.

Mental stress modifies both cholinergic neurotransmission and alternative splicing in the brain, via incompletely understood mechanisms. Here, we report that stress changes brain microRNA (miR) expression and that some of these stress-regulated miRs regulate alternative splicing. Acute and chronic immobilization stress differentially altered the expression of numerous miRs in two stress-responsive regions of the rat brain, the hippocampal CA1 region and the central nucleus of the amygdala. miR-134 and miR-183 levels both increased in the amygdala following acute stress, compared to unstressed controls. Chronic stress decreased miR-134 levels, whereas miR-183 remained unchanged in both the amygdala and CA1. Importantly, miR-134 and miR-183 share a common predicted mRNA target, encoding the splicing factor SC35. Stress was previously shown to upregulate SC35, which promotes the alternative splicing of acetylcholinesterase (AChE) from the synapse-associated isoform AChE-S to the, normally rare, soluble AChE-R protein. Knockdown of miR-183 expression increased SC35 protein levels in vitro, whereas overexpression of miR-183 reduced SC35 protein levels, suggesting a physiological role for miR-183 regulation under stress. We show stress-induced changes in miR-183 and miR-134 and suggest that, by regulating splicing factors and their targets, these changes modify both alternative splicing and cholinergic neurotransmission in the stressed brain.

The alpha7 neuronal nicotinic receptor gene (CHRNA7) has been implicated in the pathophysiology of schizophrenia by genetic and pharmacological studies. Expression of the alpha7* receptor, as measured by [(125)I]alpha-bungarotoxin autoradiography, is decreased in postmortem brain of schizophrenic subjects compared to non-mentally ill controls. Most schizophrenic patients are heavy smokers, with high levels of serum cotinine. Smoking changes the expression of multiple genes and differentially regulates gene expression in schizophrenic hippocampus. We examined the effects of smoking on CHRNA7 expression in the same tissue and find that smoking differentially regulates expression of both mRNA and protein for this gene. CHRNA7 mRNA and protein levels are significantly lower in schizophrenic nonsmokers compared to control nonsmokers and are brought to control levels in schizophrenic smokers. Sufficient protein but low surface expression of the alpha7* receptor, seen in the autoradiographic studies, suggests aberrant assembly or trafficking of the receptor.

Activation, inactivation, and desensitization are key features of ion channel behavior. We endeavor to understand these processes at the level of the single molecules and extrapolate from such microscopic models the behavior of ion channels in contexts of cellular physiology and therapeutics. In the case of ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs), it is also important to consider the nature of the dynamic changes in the chemical stimulus required for activation. The amplitude and time course of the agonist pulse provided to nAChR at a fast synapse will be vastly different from those of the ACh stimulus presented to presynaptic receptors in the brain and neither of these physiological processes will resemble the stimuli presented by nicotine self-administration or with systemic delivery of a therapeutic agent. Likewise, specific experimental protocols will provide unique stimulus profiles, which will impact the relationship between the macroscopic data and the underlying molecular processes. In this work, ion channel simulations intended to model heteromeric neuronal nAChR are conducted under varying conditions of agonist presentation, and the impact of a key microscopic process, desensitization, is studied on the macroscopic responses. With instantaneous jumps in agonist concentrations, the microscopic desensitization rate impacts essentially all aspects of the macroscopic responses, rise rates, decay rates, and both peak and steady-state currents. In contrast, with an agonist pulse like that used in Xenopus oocyte experiments, microscopic desensitization rates have a profound impact on peak current amplitude and very little effect on the kinetics of the macroscopic responses.

There have been continued efforts to develop effective antidotal therapies against poisoning with organophosphorus (OP) compounds, including nerve agents and pesticides. We reported recently that galantamine, a drug used to treat Alzheimer's disease, administered before (up to 3 h) or soon after (up to 5 min) an exposure of guinea pigs to 1.5-2 x LD50 soman or sarin effectively counteracted the acute toxicity and lethality of the nerve agents provided that the animals were also post-treated with atropine. Here, we demonstrate that administered to guinea pigs at 30 min before or up to 15 min after an acute challenge with 1 x LD50 soman, galantamine (8 mg/kg, intramuscular) alone is sufficient to counteract the lethality and acute toxicity of the nerve agent. Evidence is also provided that 100% survival can be attained when the association of appropriate doses of galantamine and atropine is administered 30-45 min after the challenge of the guinea pigs with 1 x LD50 soman. Galantamine counteracts the neurodegeneration and the changes in the nicotinic cholinergic system that result from an acute exposure of guinea pigs to 1 x LD50 soman. The results presented herein corroborate that galantamine is an effective antidote against OP poisoning.

Our previous work had shown that long-term nicotine administration improved dopaminergic markers and nicotinic receptors (nAChRs) in the striatum of monkeys with nigrostriatal damage. The present experiments were done to determine whether nicotine treatment also led to changes in the substantia nigra, the region containing dopaminergic cell bodies. Monkeys were chronically treated with nicotine in the drinking water for 6 months after which they were injected with low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine (MPTP) for a further 6-month period. Nicotine was administered until the monkeys were euthanized 2 months after the last MPTP injection. Nicotine treatment did not affect the dopamine transporter or the number of tyrosine hydroxylase positive cells in the substantia nigra of lesioned monkeys. However, nicotine administration did lead to a greater increase in alpha3/alpha6beta2* and alpha4beta2* nAChRs in lesioned monkeys compared to controls. Nicotine also significantly elevated microglia and reduced the number of extracellular neuromelanin deposits in the substantia nigra of MPTP-lesioned monkeys. These findings indicate that long-term nicotine treatment modulates expression of several molecular measures in monkey substantia nigra that may result in an improvement in nigral integrity and/or function. These observations may have therapeutic implications for Parkinson's disease.

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the alpha3, beta2, and beta4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of alpha3 and the two beta subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the alpha7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.

Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active-site gorge and a peripheral site or P-site at its mouth. Although much is known about AChE structure and the role of specific residues in catalysis, a detailed understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years, we have clarified how the P-site and A-site interact to promote catalysis. Our studies revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation of substrate hydrolysis. Because a general, nonequilibrium treatment of AChE catalysis results in complex enzyme kinetic formulations, three simpler, overlapping strategies are presented here that provide significant insights into the AChE catalytic mechanism. The strategies are (1) to choose substrates, preferably close analogs of acetylcholine, that render some intermediates in the general reaction scheme negligible; (2) obtain some of the thermodynamic parameters in this scheme with experiments that are independent of kinetic measurements.

The capability of the tertiary oximes, monoisonitrosoacetone (MINA) and diacetylmonoxime (DAM), to reactivate acetylcholinesterase (AChE) inhibited by sarin (GB) in the blood, brain, and peripheral tissues of guinea pigs was compared with that of the quaternary oximes 2-PAM, HLo7, and MMB-4. Animals were injected subcutaneously (s.c.) with 1.0 x LD(50) of GB and treated intramuscularly (i.m.) 5 min later with one of these oximes. Sixty minutes after GB exposure, tissues were collected for AChE analysis. At low doses, MINA and DAM produced significant increases in AChE activity in all brain areas examined, but no significant AChE reactivation in peripheral tissues or blood. At higher doses, MINA and DAM increased AChE activity in the brain, peripheral tissues, and blood. In contrast, the quaternary oximes produced significant reactivation in peripheral tissues and blood AChE, but no significant reactivation of brain AChE. In another study, animals were pretreated i.m. with pyridostigmine 30 min prior to s.c. challenge with 2.0 x LD(50) of GB and treated i.m. 1 min later with atropine sulfate (2.0 mg/kg), plus a varied dose of oximes. MINA and DAM prevented or terminated GB-induced seizure activity and protected against GB lethality in a dose-dependent fashion. In contrast, none of the quaternary oximes prevented or stopped GB-induced seizures. Thus, tertiary oximes reactivated AChE in the brain, improved survival, and terminated seizures following GB intoxication.

Agonist binding to Cys-loop receptors promotes a large transmembrane ion flux of several million cations or anions per second. To investigate structural bases for the dynamics (MD) simulations, X-ray crystallography, and single channel recording. MD simulations of the muscle nicotinic receptor, imbedded in a lipid bilayer with an applied transmembrane potential, reveal single cation translocation events during transient periods of channel hydration. During the simulation trajectory, cations paused for prolonged periods near several rings of anionic residues projecting from the lumen of the extracellular domain of the receptor, but subsequently the cation moved rapidly through the hydrophobic transmembrane region as the constituent alpha-helices exhibited back and forth rocking motions. Cocrystallization of acetylcholine binding protein with sulfate ions revealed coordination of five sulfates with residues from one of these charged rings; in cation-selective Cys-loop receptors this ring contains negatively charged residues, whereas in anion-selective receptors it contains positively charged residues. In the muscle nicotinic receptor, charge reversal of residues of this ring decreases unitary conductance by up to 80%. Thus in Cys-loop receptors, a series of charged rings along the ion translocation pathway concentrates hydrated ions relative to bulk solution, giving rise to charge selectivity, and then subtle motions of the hydrophobic transmembrane, coupled with transient periods of water filling, enable rapid ion flux.

Control of seizure activity is critical to survival and neuroprotection following nerve agent exposure. Extensive research has shown that three classes of drugs, muscarinic antagonists, benzodiazepines, and N-methyl-D: -aspartate antagonists, are capable of moderating these seizures. This study began to map the neural areas in rat brain that respond to these three drug classes resulting in anticonvulsant effects. Drugs of each class (scopolamine, midazolam, MK-801) were evaluated for their ability to prevent sarin-induced seizures when injected into specific brain areas (lateral ventricle, anterior piriform cortex, basolateral amygdala, area tempestas). Animals were pretreated by microinjection with saline or a dose of drug from one of the three classes 30 min prior to receiving 150 microg/kg sarin, subcutaneously, followed by 2.0 mg/kg atropine methylnitrate, intramuscularly. Animals were then returned to their cages, where electroencephalographic activity was monitored for seizures. Anticonvulsant effective doses (ED(50)) were determined using an up-down dosing procedure over successive animals. Scopolamine provided anticonvulsant effects in each area tested, while midazolam was effective in each area except the lateral ventricle. MK-801 was only effective at preventing seizures when injected into the basolateral amygdala or area tempestas. The results show a unique neuroanatomical and pharmacological specificity for control of nerve agent-induced seizures.

New compounds that target nicotinic receptors (nAChRs) have been sought to correct disorders affecting cholinergic transmission in central and peripheral synapses. A quaternary derivate of l-hyoscyamine, phenthonium (Phen), was shown by our group to enhance the spontaneous acetylcholine (ACh) release without altering the nerve-induced transmitter release at the neuromuscular junction. The effect was unrelated to membrane depolarization, and was not induced by an increase of calcium influx into the nerve terminal. Phen also presented a competitive antimuscarinic activity and blocked noncompetitively the neuromuscular transmission. In this work we re-examined the mechanisms underlying the facilitatory actions of Phen on [(3)H]-ACh release in isolated ganglia of the guinea pig ileal myenteric plexus. Exposure of the preparations to Phen (10-50 microM) increased the release of [(3)H]-ACh by 81 to 68% over the basal. The effect was not affected by the ganglionic nAChR antagonist hexamethonium (1 nM) at a concentration that inhibited the increase of [(3)H]-ACh release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP, 30 microM). Association of Phen (10 microM) with DMPP potentiated the facilitatory effect of Phen. [(3)H]-ACh release was not altered by the muscarinic antagonists atropine (1 nM) or pirenzepine (1 microM). However, both antagonists inhibited the release of [(3)H]-ACh induced by either the muscarinic M1 agonist McN-343 (10 microM) or Phen (20 microM). The facilitatory effect of Phen was not altered by CdCl(2) (50 mM), but it was potentiated in the presence of tetraethylammonium (40 mM). The results indicate that the facilitatory action of Phen appears to be mediated by an increase of the inwardly rectifying potassium channels conductance probably related to the compound antimuscarinic activity.

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.

At the request of the organizers of the XIII International Symposium on Cholinergic Mechanisms, I have selected for review three topics not well represented at the meeting: the synthesis, storage, and release of acetylcholine at cholinergic nerve endings; cholinergic-specific antigens; and neuropeptides as cholinergic co-transmitters. The first topic is illustrated by work with two model systems, the electromotor nerve terminals of the electric ray and the myenteric plexus of the guinea-pig ileum, the second by work with Chol-1, a group of cholinergic-specific gangliosides sialylated on their N-acetylgalactosamine residues, and the third by vasoactive intestinal polypeptide which is a co-transmitter at 70% of cholinergic nerve endings in brain and is also present in both of the model cholinergic systems.

The cognitive deficits seen in schizophrenia patients are likely related to abnormal glutamatergic and cholinergic neurotransmission in the prefrontal cortex. We hypothesized that these impairments may be secondary to increased levels of the astrocyte-derived metabolite kynurenic acid (KYNA), which inhibits alpha7 nicotinic acetylcholine receptors (alpha7AChR) and may thereby reduce glutamate release. Using in vivo microdialysis in unanesthetized rats, we show here that nanomolar concentrations of KYNA, infused directly or produced in situ from its bioprecursor kynurenine, significantly decrease extracellular glutamate levels in the prefrontal cortex. This effect was prevented by the systemic administration of galantamine (3 mg/kg) but not by donepezil (2 mg/kg), indicating that KYNA blocks the allosteric potentiating site of the alpha7AChR, which recognizes galantamine but not donepezil as an agonist. In separate rats, reduction of prefrontal KYNA formation by (S)-4-ethylsulfonyl benzoylalanine, a specific inhibitor of KYNA synthesis, caused a significant elevation in extracellular glutamate levels. Jointly, our results demonstrate that fluctuations in endogenous KYNA formation bidirectionally influence cortical glutamate concentrations. These findings suggest that selective attenuation of cerebral KYNA production, by increasing glutamatergic tone, might improve cognitive function in individuals with schizophrenia.