Imaging of cerebral α4ß2* nicotinic acetylcholine receptors with (-)-[(18)F]Flubatine PET: Implementation of bolus plus constant infusion and sensitivity to acetylcholine in human brain.

The positron emission tomography (PET) radioligand (-)-[(18)F]flubatine is specific to α4ß2(⁎) nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4ß2(⁎) nAChR quantification with bolus plus constant infusion (B/I) (-)-[(18)F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n=8) or B/I (n=4) administration of (-)-[(18)F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n=6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5mg over 60min beginning 5min prior to radiotracer injection); 2. A single scan B/I paradigm (n=7) lasting up to 240min with 1.5mg physostigmine administered over 60min beginning at 125min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8±3.3mL/cm(3) in thalamus, 12.9±1.6mL/cm(3) in cerebellum, and ranged from 9.8 to 12.5mL/cm(3) in other gray matter regions. The B/I paradigm with equilibrium analysis at 120min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (-)-[(18)F]flubatine VT/fP in 120min, and suggest possible sensitivity of (-)-[(18)F]flubatine binding to physostigmine-induced changes in acetylcholine levels.

Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4ß2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4ß2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4ß2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4ß2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

Impact of modulation of the α7 nicotinic acetylcholine receptor on nicotine reward in the mouse conditioned place preference test.

RATIONALE: The α7 nicotinic acetylcholine receptor (nAChR) has been implicated as a target in modulating nicotine reward. However, the effect of pharmacological agents that have been shown to alter the channel properties of the α7 nAChR is not well understood in nicotine reward. OBJECTIVES: This study aimed to investigate the impact of α7 nAChR pharmacological modulation on nicotine conditioned place preference (CPP) in mice by using positive allosteric modulators (PAMs) and a silent agonist. METHODS: The effect of the orthosteric α7 nAChR full agonist PNU282987 (1.3 and 9 mg/kg, s.c.), Type I α7 PAM NS1738 (1 and 10 mg/kg; i.p.), the Type II α7 PAM PNU120596 (0.3, 1, and 3 mg/kg, i.p.), and the α7 silent agonist NS6740 (1 and 3 mg/kg, i.p) on nicotine CPP was measured in mice. Mice were conditioned with either saline or nicotine (0.5 mg/kg) for 3 days in the CPP paradigm. RESULTS: The α7 full orthosteric agonist PNU282987 and the Type II α7 nAChR PAM PNU120596 reduced nicotine CPP, while the silent agonist NS6740 and Type I PAM NS1738 had no effect. The effects of PNU282987 and PNU120596 did not have an effect on morphine CPP. CONCLUSIONS: Taken together, our results suggest that modulation of the α7 nAChR can play important roles in nicotine CPP in mice. In addition, the Type II α7 nAChR PAM PNU120596 attenuated nicotine reward suggesting that endogenous acetylcholine/choline tone is sufficient to reduce nicotine CPP. These findings highlight a beneficial effect of using α7 nAChR PAMs in nicotine reward.

Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

The neuronal nicotinic acetylcholine receptor subunits alpha 2, alpha 3, and alpha 4 form functional receptors with the beta 2 subunit. Each of these subunit combinations shows two distinct open states (referred to as primary and secondary). The primary open states of alpha 2 beta 2, alpha 3 beta 2, and alpha 4 beta 2 receptors were 33.6 +/- 1.8 pS, 15.4 +/- 0.8 pS, and 13.3 +/- 1.5 pS, respectively. The open times of the alpha 3 beta 2 primary open state were significantly longer than the open times of the other primary conductance states. The secondary open states of alpha 2 beta 2 and alpha 3 beta 2 were 15.5 +/- 1.3 pS and 5.1 +/- 0.4 pS, respectively. Secondary open states were seen infrequently with alpha 4 beta 2. Oocytes injected with alpha 2 RNA and a 9-fold excess of beta 2 RNA showed an enhanced expression of the secondary open state.

Region-specific effects of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide on nicotine-induced increase in extracellular dopamine in vivo.

BACKGROUND AND PURPOSE: Systemic administration of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), an antagonist of nicotinic acetylcholine receptors (nAChRs) attenuated the nicotine-induced increase in dopamine levels in nucleus accumbens (NAcc). EXPERIMENTAL APPROACH: Using in vivo microdialysis, we investigated the effects of local perfusion of the novel nAChR antagonist bPiDDB into the NAcc or ventral tegmental area (VTA) on increased extracellular dopamine in NAcc, induced by systemic nicotine. We also examined the concentration-dependent effects of bPiDDB on the acetylcholine (ACh)-evoked response of specific recombinant neuronal nAChR subtypes expressed in Xenopus oocytes, using electrophysiological methods. KEY RESULTS: Nicotine (0.4 mg kg(-1), s.c.) increased extracellular dopamine in NAcc, which was attenuated by intra-VTA perfusion of mecamylamine (100 microM). Intra-VTA perfusion of bPiDDB (1 and 10 microM) reduced nicotine-induced increases in extracellular dopamine in NAcc. In contrast, intra-NAcc perfusion of bPiDDB (1 or 10 microM) failed to alter the nicotine-induced increase in dopamine in NAcc. Intra-VTA perfusion of bPiDDB alone did not alter basal dopamine levels, compared to control, nor the increased dopamine in NAcc following amphetamine (0.5 mg kg(-1), s.c.). Using Xenopus oocytes, bPiDDB (0.01-100 microM) inhibited the response to ACh on specific combinations of rat neuronal nAChR subunits, with highest potency at alpha3beta4beta3 and lowest potency at alpha6/3beta2beta3. CONCLUSIONS AND IMPLICATIONS: bPiDDB-Sensitive nAChRs involved in regulating nicotine-induced dopamine release are located in the VTA, rather than in the NAcc. As bPiDDB has properties different from the prototypical nAChR antagonist mecamylamine, further development may lead to novel nAChR antagonists for the treatment of tobacco dependence.

alpha7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents.

The functional mechanisms of noncompetitive blockade of the nicotinic acetylcholine receptor from the BC3H-1 cell line were examined using single-channel currents recorded from cell-attached patches. Channel open times were distributed as sums of two exponentials and the closed times as sums of at least four exponentials. The single-channel currents of the receptor were analyzed in terms of activation schemes in which the receptor exists in two open states and a number of closed or blocked states. The existence of two distinct open states for the acetylcholine receptor allows for predictions to be made that will distinguish between different mechanisms of blockade. Notably, predictions could be made based on the model for the sequential block of open channels, that would allow us to discriminate such a mechanism, even for ligands that appear to dissociate so slowly that sequential openings of the same channel do not appear as distinct bursts. Four noncompetitive blockers of the acetylcholine receptor were studied: tetracaine, phencyclidine, and the (+) and (-) isomers of N-allylnormetazocine (SKF-10047). All four of these ligands decreased the duration of single-channel currents without increasing the number of fast closures per burst. The data suggest that the ligands block the channel in at least two distinct ways, one of which involves a specific interaction with open channels and the other is most consistent with the blockade of channels that may be either open or closed. In addition, the duration of the open state may be allosterically lengthened by the interaction of certain blockers with another class of sites.

Muscle-type nicotinic acetylcholine receptor delta subunit determines sensitivity to noncompetitive inhibitors, while gamma subunit regulates divalent permeability.

Heterologous expression of nicotinic acetylcholine receptor (nAChR) RNAs in Xenopus oocytes was used to examine the structural basis for pharmacological and physiological differences between muscle-type and neuronal nAChRs. Neuronal nAChRs have a higher permeability to calcium than muscle-type nAChRs and display inward rectification. while muscle-type nAChRs have a linear current-voltage relation. In addition, neuronal nAChRs are more sensitive to inhibition by a class of compounds known as "ganglionic blockers". It has been shown previously that neuronal-muscle hybrid receptors show increased sensitivity to the use-dependent inhibitor of neuronal nAChRs, BTMPS, based on the presence of a neuronal beta subunit. In this study, we report that omission of gamma subunit RNA has a similar effect. alpha beta delta receptors exhibit prolonged inhibition by BTMPS; show a significant permeability to divalent ions, display inward rectification and are more sensitive to mecamylamine. However, while pharmacological effects are associated with the presence of an additional delta subunit, the physiological changes described seem to be associated with the presence or absence of a gamma subunit. These results suggest that, for nAChRs, as is also the case for non-NMDA ionotropic glutamate receptors, the crucial functional property of limiting calcium permeability can be served by a single subunit.

2-(2-Piperidyl)- and 2-(2-pyrrolidyl)chromans as nicotine agonists: synthesis and preliminary pharmacological characterization.

As part of an effort to develop a new class of subtype selective nicotine agonists, we have synthesized and tested a group of 12 hydroxylated 2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans. In rat brain membranes, all 12 compounds displayed poor affinity for [(125)I]-alpha-bunagarotoxin binding sites. In contrast, three compounds, 17c, 24, and 26, displayed moderate to high affinity for [(3)H]cytisine binding sites, while three (17b, 18b,c) and six (17a,d,e and 18a,d,e) compounds showed weak and poor affinity, respectively, for these same sites. In subsequent studies, compounds 17a and 17c were found to stimulate the efflux of (86)Rb(+) from rat cortical synaptosomes, an indication of agonist activity. Further, both 17c and 26 displayed high intrinsic activity in stimulating the release of [(3)H]dopamine from striatal synaptosomes; however, only 17c was effective at stimulating the release of [(3)H]acetylcholine from cortical synaptosomes, suggesting differential selectivity. In cloned human nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes, both 17c and 26 activated alpha7 and alpha3beta2 receptor subtypes in a dose-dependent manner, but 26 was clearly the more potent agonist. Last, neither compound displayed dose-dependent activation of alpha4beta2 nAChRs. We conclude that 2-(2-azacyclic)chromans appear to be a promising new class of nicotine agonists.

The amino terminal half of the nicotinic beta-subunit extracellular domain regulates the kinetics of inhibition by neuronal bungarotoxin.

Subtypes of nicotinic receptors previously reported to be unaffected by neuronal bungarotoxin (NBT), including alpha 3 beta 4-containing and muscle type (alpha 1 beta 1 gamma delta) receptors, are shown to be inhibited by this toxin, but with rapid kinetics of onset and recovery. This inhibition is in contrast to the slow and prolonged inhibition of alpha 3 beta 2-containing receptors, suggesting that the beta subunits determine the kinetics of NBT inhibition of alpha 3 receptors. We have coexpressed chimeric beta subunits with alpha 3, and our results show that the first 121 amino acids of the beta subunit extracellular domain are sufficient to regulate the kinetics of NBT inhibition. This domain is also an important determinant of whether cytisine will act as a full agonist or a partial agonist of nicotinic receptors formed with alpha 3.

Activation and inhibition of rat neuronal nicotinic receptors by ABT-418.

1. ABT-418 appeared to function as a relatively broad spectrum activator of neuronal nicotinic receptors, expressed in Xenopus oocytes, with little cross reactivity to the mammalian muscle receptor subtype. However, the relative potencies of ABT-418 at the various subtypes differed from those acetylcholine (ACh). For example, ACh was most potent at alpha 3 beta 2 (EC50 approximately 30 microM) and least potent at alpha 2 beta 2 (EC50 approximately 500 microM). ABT-418 was most potent at alpha 4 beta 2 and alpha 2 beta 2 (EC50 approximately 6 microM and 11 microM, respectively) and least potent at alpha 3 beta 4 (EC50 approximately 188 microM). 2. In addition to activating neuronal receptors, ABT-418 exhibited complex properties, including the inhibition of ACh responses. 3. The current responses elicited by relatively high concentrations of ABT-418 on the alpha 4 beta 2 receptor subtype were protracted beyond the application interval. The coapplication of ABT-418 with either of the use-dependent inhibitors bis(1,2,2,6,6-tetramethyl-4-pipendimyl)sebacate (BTMPS) or tetramethyl-pipenidine (TMP) eliminated the late protracted phase of the currents with only small effects on the initial activation phase. When the reversible inhibitor TMP was washed from the bath, the previously inhibited late current reappeared, suggesting that the observed mixed agonist-antagonist effects of ABT-418 and (+/-)-epibatidine on alpha 4 beta 2 were due to a concentration-dependent noncompetitive inhibition, an effect similar to that obtained for (-)-nicotine. 4. The inhibition of alpha 4 beta 2 receptors by ABT-418 was voltage-dependent. When high concentrations of ABT-418 were applied under depolarizing conditions, additional late currents could be observed under conditions which suggested that a build up of ABT-418 in an unstirred layer over the surface of the oocyte was occurring. This may have been due to the dissociation of the drug from channel blocking sites on the receptors themselves, or alternatively, from the plasma membrane of the cells.

Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain.

We show that a portion of the TM2 domain regulates the sensitivity of beta subunit-containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long-term effects of this drug on receptor function. The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long-term inhibition by nicotine. Inhibition by mecamylamine is voltage-dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane field. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis-TMP compounds is independent of voltage. Our experiments did not show any influence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory effects of nicotine are mediated by binding to a site outside the membrane's electric field. An analysis of point mutations indicates that the residues at the 6' position within the beta subunit TM2 domain may be important for determining the effects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10' position are not sufficient to produce effects, but 6' 10' double mutants show more effect than do the 6' single mutants.

Neurobiology of lithium: an update.

Lithium remains a first-line approach for the treatment of acute mania and the prophylactic management of manic-depressive illness, yet the underlying neurobiological mechanisms remain as yet undefined. In this paper we critically examine the accumulated preclinical and clinical evidence for the action of lithium in the brain and suggest areas that may be most productive for future investigation, i.e., membrane transport systems, neurotransmitter receptor regulation, second messenger generating systems, protein kinase C (PKC) regulation, and gene expression. In their experimental design, preclinical investigations have often jeopardized the physiologic relevance of their studies by a relative lack of attention to issues such as therapeutic concentrations, acute versus chronic exposure, and a lack of adequate cation and/or psychotropic controls. Future studies should account for the established prophylactic efficacy of lithium, the higher risk for relapse into mania after abrupt discontinuation, the ability of lithium to stabilize recurrent depression associated with unipolar disorder, and the efficacy of lithium in the treatment of refractory major depressive disorder in the presence of an antidepressant. Studies of the action of lithium in receptor mediated phosphoinositide signaling in the brain over the past several years have opened up heuristic lines of investigation that stem from lithium's uncompetitive inhibition of the enzyme inositol monophosphatase. Subsequent studies involving regulation of inositol transport, PKC isozymes and activity, and the expression of the major PKC substrate MARCKS (myristoylated alanine-rich C-kinase substrate) have offered potential avenues for understanding the complexity of the action of long-term lithium in the brain. These studies will offer us a better understanding of the neuroanatomical sites of action of lithium and together with ongoing clinical investigations using brain imaging in patients with manic-depressive illness a more complete understanding of the pathophysiology of this disease.

Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells.

The alpha7 nicotinic receptor partial agonist DMXB protected differentiated PC12 cells from NGF+ serum deprivation over a concentration range (1-10 microM) that correlated with activation of protein kinase C. Increased toxicity was observed at a higher concentration of DMXB (30 microM) that did not elevate protein kinase C activity, but did increase tyrosine protein kinase activity. Neuroprotection was blocked with the protein kinase C-inhibitor bis-indolemaleimide, while toxicity was attenuated with the tyrosine protein kinase-antagonists herbimycin and genistein. The alpha7-selective antagonist methyllyconitine attenuated both the protective and toxic actions of DMXB, but in temporally distinct manners. Methyllyconitine (1 microM) attenuated toxicity when added 10 s before, but not 10 s after, 30 microM DMXB. In contrast, it blocked neuroprotection when added 10 min post-agonist addition. This temporal difference in receptor-activation that was necessary for protection vs. toxicity reflected the time courses for agonist-induced desensitization of the receptor expressed in Xenopus oocytes. These results indicate that alpha7 nicotinic receptors act through different intracellular transduction processes to protect or kill cells. Further, they suggest that the transduction processes may be differentially activated depending on the amplitude and duration of calcium signals.

3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat alpha7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner.

The alpha7 nicotinic receptor agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB; GTS-21) was investigated for its ability to: (1) activate a variety of nicotinic receptor subtypes in Xenopus oocytes; (2) improve passive avoidance and spatial Morris water task performances in mecamylamine-sensitive manners in bilaterally nucleus basalis lesioned rats; and (3) elevate high-affinity [3H]acetylcholine (ACh) and high-affinity alpha-[125I]bungarotoxin binding in rat neocortex following 2 weeks of daily injections. DMXB (100 microM) activated alpha7 homo-oligomeric receptors, without significant activity at alpha2-, alpha3- and alpha4-containing subtypes. Mecamylamine blocked rat alpha7 receptors weakly if co-administered with agonist, but much more potently when pre-applied. Bilateral ibotenic acid lesions of the nucleus basalis interfered with passive avoidance and spatial memory-related behaviors. DMXB (0.5 mg/kg, i.p.) improved passive avoidance behavior in lesioned animals in a mecamylamine-sensitive manner. DMXB (0.5 mg/kg 15 min before each session) also improved performance in the training and probe components of the Morris water task. DMXB-induced improvement in the probe component but not the training phase was mecamylamine-sensitive. [3H]ACh binding was elevated after 14 days of daily i.p. injections with 0.2 mg/kg nicotine but not after 1 mg/kg DMXB. Neither drug elevated high-affinity alpha-[125I]bungarorotoxin binding over this interval.

alpha7 receptor-selective agonists and modes of alpha7 receptor activation.

The alpha7-selective agonists 3-(2, 4-dimethoxybenzylidene)-anabaseine (GTS-21), also known as DMXB, and 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) produce a variety of behavioral and cytoprotective effects that may be related to the activation of either large transient currents at high concentrations or small sustained currents at lower agonist concentrations. We are using acutely dissociated hypothalamic neurons, which express a central nervous system (CNS) alpha7-type receptor, to test a model for the concentration-dependent desensitization of alpha7-mediated responses. Our results confirm that 4OH-GTS-21 is a potent activator of neuronal alpha7 nicotinic-acetylcholine receptor. The rapid application of agonist leads to a brief period of maximal receptor-activation followed by desensitization. Rise rates, decay rates, and the degree to which current was desensitized were all concentration-dependent. Following the initial peak response to a 300-microM 4OH-GTS-21 application, current is reduced to baseline values within about 100 ms. Application of 30 microM 4OH-GTS-21 produced both a transient peak current and a sustained current that decayed only slowly after the removal of agonist. In the case of a 300-microM 4OH-GTS-21 application, after agonist was removed, we saw a rebound response up to the level of the 30-microM sustained current. The data, therefore, suggest that a sufficient level of agonist occupation can be retained on the receptor to promote activation for up to several hundred milliseconds.

Lithium modulates desensitization of the glutamate receptor subtype gluR3 in Xenopus oocytes.

Analysis of splice variants and site-directed mutants of the AMPA receptor GluR3 expressed in Xenopus oocytes has shown that lithium produces a large potentiation of the GluR3 flop splice variant and suggested that lithium might inhibit rapid desensitization, which is characteristic of this receptor (Karkanias, N. and Papke, R., Subtype-specific effects of lithium on glutamate receptor function. J. Neurophysiol., 81 (1999) 1506-1512). We now show that mutation of the 769R/ G desensitization site (Lomeli, H.M.J., Melcher, T., Hoger, T., Geiger, J.R., Kuner, T., Monyer, H., Higuchi, M.B.A. and Seeburg, P.H, Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. Science, 9(266) (1994) 1709-1713) greatly attenuates the lithium-induced potentiation of GluR3. Additionally, experiments with the non-desensitizing site-directed mutant GluR3(L507Y) (Stern-Bach, Y., Russo, S., Neuman, M. and Rosenmund, C., A point mutation in the glutamate binding site blocks desensitization of AMPA receptors. Neuron, 21 (1998) 907-918) further confirms that lithium enhances GluR3 responses by reducing desensitization, since lithium's effects are reversed in this mutant. Lithium's effects on GluR3 desensitization are distinct from the effects of aniracetam on desensitization. Specifically, aniracetam, which potentiates wild-type AMPA receptors, is ineffective on the non-desensitizing GluR3(L507Y) mutant, but has synergistic effects with lithium on wild-type receptors.

The correction of alpha7 nicotinic acetylcholine receptor concentration-response relationships in Xenopus oocytes.

The rapid desensitization of alpha7 nicotinic acetylcholine receptors (nAChR) has presented a serious problem for the characterization of this receptor subtype, potentially confounding the interpretation of concentration-response relationships. However, the consistency of cell geometry and solution flow in oocyte recordings permits estimations of instantaneous concentrations to be made in this system. Results interpreted with predicted instantaneous concentrations suggest that estimates of EC50 derived from conventional analysis may overestimate the actual EC50 values by a factor of 10 and underestimate Hill slopes by a factor of 2-3. If the limiting desensitization process of alpha7 receptors is driven by the agonist concentration itself rather than by time-dependent processes, then similar dependencies may exist between the response and instantaneous agonist concentration in other systems.

An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the alpha 7 subtype.

The agonist properties of acetylcholine (ACh), tetramethylammonium, ethyl-trimethylammonium and choline were evaluated for muscle and neuronal nicotinic receptors in Xenopus oocytes. The only essential feature for a neuronal receptor agonist appears to be the charged nitrogen. For specific receptor subtypes, other structural elements appear permissive (neither increasing nor decreasing activity) or non-permissive (decreasing activity). Choline was a full agonist for alpha 7, but a hydroxyl group was strongly non-permissive for other receptor subtypes (alpha 1 beta 1 gamma delta, alpha 3 beta 4, alpha 3 beta 2, and alpha 4 beta 2). The binding of these ligands to brain membranes is consistent with the electrophysiological results. Physiological concentrations of choline desensitize alpha 7 receptors to ACh suggesting that, in vivo, choline may regulate both the activation and inactivation of this receptor.

Characterization of nicotinic acetylcholine receptor channels of the TE671 human medulloblastoma clonal line.

Acetylcholine (ACh)-gated single channel events were studied on the TE671 human medulloblastoma clonal cell line by the use of the cell-attached patch clamp technique. Channel activity was detected (86% probability) in the presence of 0.1-2 microM ACh but not (0% probability) in the absence of agonist or in the presence of 1 microM alpha-bungarotoxin (Bgt). This effect of Bgt was reversible within 1 h. The most prominent channel type had a conductance of 50 pS. The kinetics of opening and closing of the channel were similar to that for skeletal muscle nicotinic acetylcholine receptors.