Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.

Event Abstract Back to Event Serotonin induces central fatigue by inhibiting action potential initiation in motoneurons. Florence Cotel1, 2*, Richard Exley3, Stephanie Cragg3 and Jean-François M. Perrier2 1 Queensland Brain Institute, Australia 2 University of Copenhagen, Department of Neuroscience and Pharmacology, Denmark 3 University of Oxford, Department of Physiology, Anatomy and Genetics, United Kingdom Neuronal output classically refers to the creation of action potentials at the axon initial segment that propagate along the axon to induce neurotransmitter release at the terminals, and, thereby, mediate a communication with postsynaptic partners. Hence information transfer critically relies on the initiation of spikes. In a slice preparation from the spinal cord of the adult turtle, we found that activation of serotonergic receptors 5-HT1A (5-HT1AR), specifically located at the axon initial segment (AIS), dampened spike initiation in motoneurons by inhibiting the Na+ channels responsible for action potential genesis [1-3]. This induced an increase of the action potential threshold and in some instances a failure of its propagation. We then tested if synaptic release of serotonin (5-HT) could activate the mechanism we uncovered. Immunohistochemical staining indicated that the somatodendritic membrane of motoneurons is densely innervated by serotonergic synaptic boutons. By contrast, the AIS is devoid of 5-HT terminals, suggesting that extrasynaptic 5-HT1AR in this compartment are activated by spillover of 5-HT. Indeed prolonged stimulations of the serotonergic raphe-spinal pathway increased extracellular 5-HT in the vicinity of the AIS to a concentration sufficient to activate 5-HT1AR and decreased the excitability of motoneurons. Raphe neurons activity level correlates with motor exercise. We showed that increased release of serotonin, as what occurs during intense motor activity, downregulated motoneuron excitability. This represents the first cellular mechanism for central fatigue and confirms many indirect evidence that serotonin is involved in the phenomenon. In conclusion the experimental evidence of a serotonergic modulation of action potential genesis at the AIS of motoneurons, that mediates central fatigue, highlights the functional importance of the regulation of spike initiation. Acknowledgements The project was funded by Danish Medical Research council Grant 09-123456, Lundbeckfonden, Owensenske Fond, Agnes and Poul Friis Fond, Association Française contre les Myopathies and Antidoping Danmark. The participation to the workshop was supported by a CNS 2015 travel award. References [1] F. Cotel, R. Exley, S.J. Cragg, and J.F. Perrier, Serotonin spillover onto the axon initial segment of motoneurons induces central fatigue by inhibiting action potential initiation. Proc Natl Acad Sci U S A 110 (2013) 4774-9. [2] J.F. Perrier, and F. Cotel, Serotonin differentially modulates the intrinsic properties of spinal motoneurons from the adult turtle. J Physiol 586 (2008) 1233-8. [3] J.F. Perrier, and F. Cotel, Serotonergic modulation of spinal motor control. Curr Opin Neurobiol 33C (2014) 1-7. Keywords: spike initiation, motoneurons, central fatigue, Serotonin, spillover Conference: Spike Initiation: Models & Experiments, Prague, Czechia, 22 Jul - 22 Jul, 2015. Presentation Type: Oral Presentation Topic: Neuroscience Citation: Cotel F, Exley R, Cragg S and Perrier JM (2015). Serotonin induces central fatigue by inhibiting action potential initiation in motoneurons.. Front. Neurosci. Conference Abstract: Spike Initiation: Models & Experiments. doi: 10.3389/conf.fnins.2015.90.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Jul 2015; Published Online: 06 Jul 2015. * Correspondence: Dr. Florence Cotel, Queensland Brain Institute, St Lucia, Queensland, 4072, Australia, f.cotel@uq.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Florence Cotel Richard Exley Stephanie Cragg Jean-François M Perrier Google Florence Cotel Richard Exley Stephanie Cragg Jean-François M Perrier Google Scholar Florence Cotel Richard Exley Stephanie Cragg Jean-François M Perrier PubMed Florence Cotel Richard Exley Stephanie Cragg Jean-François M Perrier Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chronic nicotine exposure gives rise to neural adaptations that change whole cell physiology and behaviour mainly by interacting with neuronal nicotinic acetylcholine receptors (nAChRs).The major nicotine-induced neuroadaptation is the up-regulation of brain nAChRs by means of cell-delimited post-translational mechanisms. We review what is known of the processes regulating nAChR assembly, degradation and trafficking, and how nicotine-induced modulation of these processes leads to nAChR up-regulation and changes in downstream neuronal plasticity at molecular, cellular and circuit level.

Nicotine directly regulates striatal dopamine (DA) neurotransmission via presynaptic nicotinic acetylcholine receptors (nAChRs) that are α6β2 and/or α4β2 subunit-containing, depending on region. Chronic nicotine exposure in smokers upregulates striatal nAChR density, with some reports suggesting differential impact on α6- or α4-containing nAChRs. Here, we explored whether chronic nicotine exposure modifies striatal DA transmission, whether the effects of acute nicotine on DA release probability persist and whether there are modifications to the regulation of DA release by α6-subunit-containing (*) relative to non-α6* nAChRs in nucleus accumbens (NAc) and in caudate-putamen (CPu). We detected electrically evoked DA release at carbon-fiber microelectrodes in striatal slices from mice exposed for 4-8 weeks to nicotine (200 μg/mL in saccharin-sweetened drinking water) or a control saccharin solution. Chronic nicotine exposure subtly reduced striatal DA release evoked by single electrical pulses, and in NAc enhanced the range of DA release evoked by different frequencies. Effects of acute nicotine (500 nm) on DA release probability and its sensitivity to activity were apparent. However, in NAc there was downregulation of the functional dominance of α6-nAChRs (α6α4β2β3), and an emergence in function of non-α6* nAChRs. In CPu, there was no change in the control of DA release by its α6 nAChRs (α6β2β3) relative to non-α6. These data suggest that chronic nicotine subtly modifies the regulation of DA transmission, which, in NAc, is through downregulation of function of a susceptible population of α6α4β2β3 nAChRs. This imbalance in function of α6:non-α6 nAChRs might contribute to DA dysregulation in nicotine addiction.

Motor fatigue induced by physical activity is an everyday experience characterized by a decreased capacity to generate motor force. Factors in both muscles and the central nervous system are involved. The central component of fatigue modulates the ability of motoneurons to activate muscle adequately independently of the muscle physiology. Indirect evidence indicates that central fatigue is caused by serotonin (5-HT), but the cellular mechanisms are unknown. In a slice preparation from the spinal cord of the adult turtle, we found that prolonged stimulation of the raphe-spinal pathway-as during motor exercise-activated 5-HT1A receptors that decreased motoneuronal excitability. Electrophysiological tests combined with pharmacology showed that focal activation of 5-HT1A receptors at the axon initial segment (AIS), but not on other motoneuronal compartments, inhibited the action potential initiation by modulating a Na+ current. Immunohistochemical staining against 5-HT revealed a high-density innervation of 5-HT terminals on the somatodendritic membrane and a complete absence on the AIS. This observation raised the hypothesis that a 5-HT spillover activates receptors at this latter compartment. We tested it by measuring the level of extracellular 5-HT with cyclic voltammetry and found that prolonged stimulations of the raphe-spinal pathway increased the level of 5-HT to a concentration sufficient to activate 5-HT1A receptors. Together our results demonstrate that prolonged release of 5-HT during motor activity spills over from its release sites to the AIS of motoneurons. Here, activated 5-HT1A receptors inhibit firing and, thereby, muscle contraction. Hence, this is a cellular mechanism for central fatigue.

Polymorphisms in the gene for the α5 nicotinic acetylcholine receptor (nAChR) subunit are associated with vulnerability to nicotine addiction. However, the underlying normal functions of α5-containing nAChRs in the brain are poorly understood. Striatal dopamine (DA) transmission is critical to the acquisition and maintenance of drug addiction and is modulated strongly by nicotine acting at heteromeric β2-containing (β2*) nAChRs. We explored whether α5 subunits, as well as α4, α6, and β3 subunits, participate in the powerful regulation of DA release probability by β2* nAChRs in nucleus accumbens (NAc) core and in dorsal striatum [caudatoputamen (CPu)]. We detected evoked dopamine release using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in striatal slices from mice with deletions of α4, α5, α6, or β3 subunits. We show that the nAChR subtypes that dominantly regulate dopamine transmission depend critically upon α5 subunits in the dorsal CPu in α4α5(non-α6)β2-nAChRs but not in NAc core, where α4α6β2β3-nAChRs are required. These data reveal the distinct populations of nAChRs that govern DA transmission in NAc core versus dorsal CPu. Furthermore, they indicate that α5 subunits are critical to the regulation of DA transmission by α4β2* nAChRs in regions of striatum associated with habitual and instrumental responses (dorsal CPu) rather than pavlovian associations (NAc).

Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit α4- and subunit α6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the α4 subunit, but not the α6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits α4 and α6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that α4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing α4 and α6 subunits, underlies nicotine self-administration and its long-term maintenance.

Striatal dopamine (DA) and acetylcholine (ACh) regulate motivated behaviors and striatal plasticity. Interactions between these neurotransmitters may be important, through synchronous changes in parent neuron activities and reciprocal presynaptic regulation of release. How DA signaling is regulated by striatal muscarinic receptors (mAChRs) is unresolved; contradictory reports indicate suppression or facilitation, implicating several mAChR subtypes on various neurons. We investigated whether mAChR regulation of DA signaling varies with presynaptic activity and identified the mAChRs responsible in sensorimotor- versus limbic-associated striatum. We detected DA in real time at carbon fiber microelectrodes in mouse striatal slices. Broad-spectrum mAChR agonists [oxotremorine-M, APET (arecaidine propargyl ester tosylate)] decreased DA release evoked by low-frequency stimuli (1–10 Hz, four pulses) but increased the sensitivity of DA release to presynaptic activity, even enhancing release by high frequencies (e.g., >25 Hz for four pulses). These bidirectional effects depended on ACh input to striatal nicotinic receptors (nAChRs) on DA axons but not GABA or glutamate input. In caudate–putamen (CPu), knock-out of M 2 - or M 4 -mAChRs (not M 5 ) prevented mAChR control of DA, indicating that M 2 - and M 4 -mAChRs are required. In nucleus accumbens (NAc) core or shell, mAChR function was prevented in M 4 -knock-outs, but not M 2 - or M 5 -knock-outs. These data indicate that striatal mAChRs, by inhibiting ACh release from cholinergic interneurons and thus modifying nAChR activity, offer variable control of DA release probability that promotes how DA release reflects activation of dopaminergic axons. Furthermore, different coupling of striatal M 2 /M 4 -mAChRs to the control of DA release in CPu versus NAc suggests targets to influence DA/ACh function differentially between striatal domains.

Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface-type recording chambers, they have been harder to obtain in submerged-type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch-clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave-ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.

Striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Both nicotine and endogenous acetylcholine (ACh) regulate striatal DA release via β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) on striatal axons. The subfamily of β2*-nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of the α6β2*-nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real time using fast-scan cyclic voltammetry at carbon-fibre microelectrodes in mouse striatal slices. We show that α6β2*-nAChRs dominate in the control of DA release by ACh and nicotine in NAc but have a more minor role in CPu alongside other β2*-nAChRs (e.g. α4*). These data offer new insights to suggest striatal α6*-nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.

The substantia nigra pars reticulata (SNr) forms a principal output from the basal ganglia. It also receives significant histamine (HA) input from the tuberomammillary nucleus whose functions in SNr remain poorly understood. One identified role is the regulation of serotonin (5-HT) neurotransmission via the HA-H-3 receptor. Here we have explored regulation by another HA receptor expressed in SNr, the H-2-receptor (H2R), by monitoring electrically evoked 5-HT release with fast-scan cyclic voltammetry at carbon-fiber microelectrodes in SNr in rat brain slices. Selective H2R antagonists (inverse agonists) ranitidine and tiotidine enhanced 5-HT release while the agonist amthamine suppressed release. The 'neutral' competitive antagonist burimamide alone was without effect but prevented ranitidine actions indicating that inverse agonist effects result from constitutive H2R activity independent of HA tone. H2R control of 5-HT release was most apparent (from inverse agonist effects) at lower frequencies of depolarization (<= 20 Hz), and prevailed in the presence of antagonists of GABA, glutamate or H-3-HA receptors. These data reveal that H(2)Rs in SNr are constitutively active and inhibit 5-HT release through H(2)Rs on 5-HT axons. These data may have therapeutic implications for Parkinson's disease, when SNr HA levels increase, and for neuropsychiatric disorders in which 5-HT is pivotal.

The effects of nicotine on dopamine transmission from mesostriatal dopamine neurons are central to its reinforcing properties. Only recently however, has the influence of presynaptic nicotinic receptors (nAChRs) on dopaminergic axon terminals within striatum begun to be understood. Here, rather than simply enhancing ( or inhibiting) dopamine release, nAChRs perform the role of a presynaptic filter, whose influence on dopamine release probability depends on presynaptic activity in dopaminergic as well as cholinergic neurons. Both mesostriatal dopaminergic neurons and striatal cholinergic interneurons play key roles in motivational and sensorimotor processing by the basal ganglia. Moreover, it appears that the striatal influence of dopamine and ACh cannot be fully appreciated without an understanding of their reciprocal interactions. We will review the powerful filtering by nAChRs of striatal dopamine release and discuss its dependence on activity in dopaminergic and cholinergic neurons. We will also review how nicotine, acting via nAChR desensitization, promotes the sensitivity of dopamine synapses to activity. This filtering action might provide a mechanism through which nicotine promotes how burst activity in dopamine neurons facilitates goal-directed behaviour and reinforcement processing. More generally, it indicates that we should not restrict our view of presynaptic nAChRs to simply enhancing neurotransmitter release. We will also summarize current understanding of the forms and functions of the diverse nAChRs purported to exist on dopaminergic axons. A greater understanding of nAChR form and function is imperative to guide the design of ligands with subtype-selective efficacy for improved therapeutic interventions in nicotine addiction as well as Parkinson's disease.

Modulation of striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Nicotine, by desensitizing β 2 subunit-containing ( β 2 * ) nicotinic acetylcholine receptors (nAChRs) on striatal DA axons, significantly enhances how DA is released by reward-related burst activity compared to nonreward-related tonic activity. This action provides a synaptic mechanism for nicotine to facilitate the DA-dependent reinforcement. The subfamily of β 2 * -nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of α 6 β 2 * -nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real-time using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in mouse striatal slices. The α 6-specific antagonist α -conotoxin-MII suppressed DA release evoked by single and low-frequency action potentials and concurrently enhanced release by high-frequency bursts in a manner similar to the β 2 * -selective antagonist dihydro- β -erythroidine (DH β E) in NAc, but less so in CPu. The greater role for α 6 * -nAChRs in NAc was not due to any confounding regional difference in ACh tone since elevated ACh levels (after the acetylcholinesterase inhibitor ambenonium) had similar outcomes in NAc and CPu. Rather, there appear to be underlying differences in nAChR subtype function in NAc and CPu. In summary, we reveal that α 6 β 2 * -nAChRs dominate the effects of nicotine on DA release in NAc, whereas in CPu their role is minor alongside other β 2 * -nAChRs (eg α 4 * ), These data offer new insights to suggest striatal α 6 * -nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.

Effects of derivatives of coclaurine (C), which mimic the ‘eastern’ or the nonquaternary halves of the alkaloids tetrandrine or d‐tubocurarine, respectively, both of which are inhibitors of nicotinic acetylcholine receptors (nACh), were examined on recombinant, human α7, α4β2 and α4β4 nACh receptors expressed in Xenopus oocytes and clonal cell lines using two‐electrode voltage clamping and radioligand binding techniques. In this limited series, Cs have higher affinity and are most potent at α4 subunit‐containing‐nACh receptors and least potent at homomeric α7 receptors, and this trend is very marked for the N‐unsubstituted C and its O,O′‐bisbenzyl derivative. 7‐O‐Benzyl‐N‐methylcoclaurine (BBCM) and its 12‐O‐methyl derivative showed the highest affinities and potencies at all three receptor subtypes, and this suggests that lipophilicity at C7 and/or C12 increases potency. Laudanosine and armepavine (A) were noncompetitive and voltage‐dependent inhibitors of α7, α4β2 or α4β4 receptors, but the bulkier C7‐benzylated 7BNMC (7‐O‐benzyl‐N‐methylcoclaurine) and 7B12MNMC (7‐O‐benzyl‐N,12‐O‐dimethyl coclaurine) were voltage‐independent, noncompetitive inhibitors of nACh receptors. Voltage‐dependence was also lost on going from A to its N‐ethyl analogue. These studies suggest that C derivatives may be useful tools for studies characterising the antagonist and ion channel sites on human α7, α4β2 or α4β4 nACh receptors and for revealing structure–function relationships for nACh receptor antagonists. British Journal of Pharmacology (2005) 146, 15–24. doi:10.1038/sj.bjp.0706307

Cytisine (cy) is a potent and competitive partial agonist at alpha4 subunit-containing nicotinic acetylcholine (nACh) receptors while at homomeric alpha7-nACh receptors it behaves as a full agonist with a relatively lower potency. In the present study, we assessed the effects of bromination or iodination of the pyridone ring of cy and N-methylcytisine (N-Me-cy) on the effects of these compounds on recombinant human (h) alpha7, halpha4beta2 and halpha4beta4 nACh receptors expressed in clonal cell lines and Xenopus oocytes. Halogenation at C(3) of cy or N-Me-cy usually brings about a marked increase in both affinity and efficacy at halpha7, halpha4beta2 and halpha4beta4 nACh, the extent of which depends on whether the halogen is bromine or iodine, and upon receptor subtype. The effects of halogenation at C(5) are strongly influenced by the specific halogen substituent so that bromination causes a decrease in both affinity and efficacy while iodination decreases affinity but its effects on efficacy range from a decrease (halpha7, halpha4beta4 nACh receptors) to a marked increase (halpha4beta2 nACh receptors). Based on these findings, which differ from those showing that neither the affinity nor efficacy of nicotine, 3-(2-azetidinylmethoxy)-pyridine or epibatidine are greatly affected by halogenation, dehalogenation or halogen exchange at equivalent positions, we suggest that cy, N-Me-cy and their halo-isosteres bind to neuronal nACh receptors in a different orientation allowing the halogen atom to interact with a hydrophobic halogen-accepting region within the predominantly hydrophobic agonist-binding pocket of the receptors.