Highly Selective and Potent α4ß2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats.

The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

Pharmacokinetics and pharmacodynamics of oral mecamylamine - development of a nicotinic acetylcholine receptor antagonist cognitive challenge test using modelling and simulation.

A pharmacologic challenge model with a nicotinic antagonist could be an important tool not only to understand the complex role of the nicotinic cholinergic system in cognition, but also to develop novel compounds acting on the nicotinic acetylcholine receptor. The objective was to develop a pharmacokinetic-pharmacodynamic (PKPD) model using nonlinear mixed effects (NLME) methods to quantitate the pharmacokinetics of three oral mecamylamine doses (10, 20 and 30 mg) and correlate the plasma concentrations to the pharmacodynamic effects on a cognitive and neurophysiologic battery of tests in healthy subjects. A one-compartment linear kinetic model best described the plasma concentrations of mecamylamine. Mecamylamine's estimated clearance was 0.28 ± 0.015 L min-1. The peripheral volume of distribution (291 ± 5.15 L) was directly related to total body weight. Mecamylamine impaired the accuracy and increased the reaction time in tests evaluating short term working memory with a steep increase in the concentration-effect relationship at plasma concentrations below 100 µg L-1. On the other hand, mecamylamine induced a decrease in performance of tests evaluating visual and fine motor coordination at higher plasma concentrations (EC50 97 µg L-1). Systolic and diastolic blood pressure decreased exponentially after a plasma mecamylamine concentration of 80 µg L-1, a known effect previously poorly studied in healthy subjects. The developed mecamylamine PKPD model was used to quantify the effects of nicotinic blockade in a set of neurophysiological tests in humans with the goal to provide insight into the physiology and pharmacology of the nicotinic system in humans and the possibility to optimize future trials that use mecamylamine as a pharmacological challenge.

Abuse liability assessment of an e-cigarette refill liquid using intracranial self-stimulation and self-administration models in rats.

BACKGROUND: The popularity of electronic cigarettes (ECs) has increased dramatically despite their unknown health consequences. Because the abuse liability of ECs is one of the leading concerns of the Food and Drug Administration (FDA), models to assess it are urgently needed to inform FDA regulatory decisions regarding these products. The purpose of this study was to assess the relative abuse liability of an EC liquid compared to nicotine alone in rats. Because this EC liquid contains non-nicotine constituents that may enhance its abuse liability, we hypothesized that it would have greater abuse liability than nicotine alone. METHODS: Nicotine alone and nicotine dose-equivalent concentrations of EC liquid were compared in terms of their acute effects on intracranial self-stimulation (ICSS) thresholds, acquisition of self-administration, reinforcing efficacy (i.e., elasticity of demand), blockade of these behavioral effects by mecamylamine, nicotine pharmacokinetics and nicotinic acetylcholine receptor binding and activation. RESULTS: There were no significant differences between formulations on any measure, except that EC liquid produced less of an elevation in ICSS thresholds at high nicotine doses. CONCLUSIONS: Collectively, these findings suggest that the relative abuse liability of this EC liquid is similar to that of nicotine alone in terms of its reinforcing and reinforcement-enhancing effects, but that it may have less aversive/anhedonic effects at high doses. The present methods may be useful for assessing the abuse liability of other ECs to inform potential FDA regulation of those products.

Enhanced brain penetration of hexamethonium in complexes with derivatives of fullerene C60.

The present report describes development of hexamethonium complexes based on fullerene C60. Hexamethonium has a limited penetration into CNS and therefore can antagonize central effects of nicotine only when given at high doses. In the present studies conducted in laboratory rodents, intraperitoneal administration of hexamethonium-fullerene complexes blocked effects of nicotine (convulsions and locomotor stimulation). When compared to equimolar doses of hexamethonium, complexes of hexamethonium with derivatives of fullerene C60 were 40 times more potent indicating an enhanced ability to interact with central nicotine receptors. Thus, fullerene C60 derivatives should be explored further as potential carrier systems for polar drug delivery into CNS.

Pharmacokinetics of cytisine, an α4 ß2 nicotinic receptor partial agonist, in healthy smokers following a single dose.

Cytisine, an α4 ß2 nicotinic receptor partial agonist, is a plant alkaloid that is commercially extracted for use as a smoking cessation medication. Despite its long history of use, there is very little understanding of the pharmacokinetics of cytisine. To date, no previous studies have reported cytisine concentrations in humans following its use as a smoking cessation agent. A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated for analysis of Tabex® and nicotine-free oral strips, two commercial products containing cytisine. A sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single-dose pharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3 mg oral dose administration of cytisine. Cytisine was detected in all plasma samples collected after administration, including 15 min post-dose and at 24 h. Cytisine was renally excreted and detected as an unchanged drug. No metabolites were detected in plasma or urine collected in the study. No adverse reactions were reported.

Discovery of a novel alpha-7 nicotinic acetylcholine receptor agonist series and characterization of the potent, selective, and orally efficacious agonist 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (SEN15924, WAY-361789).

Alpha-7 nicotinic acetylcholine receptors (α7 nAChR) are implicated in the modulation of many cognitive functions such as attention, working memory, and episodic memory. For this reason, α7 nAChR agonists represent promising therapeutic candidates for the treatment of cognitive impairment associated with Alzheimer's disease (AD) and schizophrenia. A medicinal chemistry effort, around our previously reported chemical series, permitted the discovery of a novel class of α7 nAChR agonists with improved selectivity, in particular against the α3 receptor subtype and better ADME profile. The exploration of this series led to the identification of 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (25, SEN15924, WAY-361789), a novel, full agonist of the α7 nAChR that was evaluated in vitro and in vivo. Compound 25 proved to be potent and selective, and it demonstrated a fair pharmacokinetic profile accompanied by efficacy in rodent behavioral cognition models (novel object recognition and auditory sensory gating).

N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity.

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.

Negative allosteric modulation of nicotinic acetylcholine receptors blocks nicotine self-administration in rats.

Drugs that antagonize nicotinic acetylcholine receptors (nAChRs) can be used to inhibit nicotine-induced behavior in both humans and animals. The aim of our experiments is to establish a proof-of-principle that antagonism of nAChRs by negative allosteric modulation can alter behavior in a relevant animal model of addiction, nicotine self-administration. We have identified a novel, negative allosteric modulator of nAChRs, UCI-30002 [N-(1,2,3,4-tetrahydro-1-naphthyl)-4-nitroaniline], with selectivity for the major neuronal nAChR subtypes over muscle-type nAChRs. After systemic administration, UCI-30002 significantly reduces nicotine self-administration in rats on both fixed ratio and progressive ratio schedules of reinforcement. The minimum effective dose that significantly alters nicotine self-administration corresponds to brain concentrations of UCI-30002 that produce at least 30% inhibition of the major neuronal nAChR subtypes measured in vitro. UCI-30002 has no effect on responding for food reinforcement in rats on either type of schedule, indicating that there is no effect on general responding or natural reward. UCI-30002 represents validation of the concept that negative allosteric modulators may have significant benefits as a strategy for treating nicotine addiction and encourages the development of subtype-selective modulators.

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD).

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N,N'-bis-nicotinium analogs, affording a lead compound, N,N'-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC(50)=500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 microM) inhibited the response to acetylcholine at alpha3beta4, alpha4beta4, alpha4beta2, and alpha1beta1varepsilondelta receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood-brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Nicotine partially protects against paraquat-induced nigrostriatal damage in mice; link to alpha6beta2* nAChRs.

Epidemiological studies indicate that smoking is a negative, and exposure to pesticides, a positive risk factor for Parkinson's disease (PD). The purpose of this study was to assess the interplay between these two factors in a rodent model of nigrostriatal damage. To approach this, mice were administered nicotine, the agent in smoke implicated in neuroprotection. They were then treated for 3 weeks with the pesticide, paraquat, while nicotine was continued. Paraquat treatment decreased (25%) nigral dopaminergic neurons, consistent with previous results. Chronic nicotine administration significantly protected against nigral cell damage, with only a 16% decline in mice treated with both nicotine and paraquat. Paraquat treatment also decreased (14%) the striatal dopamine transporter, an effect that was partially prevented by nicotine. These changes in the striatal dopamine transporter paralleled those in a select striatal alpha6beta2* nicotinic receptor (nAChR) subtype. In contrast, striatal alpha4beta2* nAChRs were not decreased with paraquat treatment, suggesting they are on a differential subset of dopaminergic terminals. The results show that nicotine treatment partially protects against paraquat-induced declines in nigrostriatal dopaminergic neurons to which a select population of alpha6beta2* nAChRs are localized. Moreover, these data support epidemiological findings that environmental influences can elicit opposing effects on nigrostriatal dopaminergic integrity.

A novel nicotinic acetylcholine receptor antagonist radioligand for PET studies.

Using positron emission tomography (PET) with a specific and selective radioligand targeting nicotinic acetylcholine receptor (nAChR) would allow us to better understand various nAChR related CNS disorders. The use of radiolabeled nAChR antagonists would provide a much safer pharmacological profile, avoiding most peripheral side effects that might be generated from radiolabeled nAChR agonists even at the tracer level; thus, PET imaging with nAChR antagonists would facilitate clinical application. A potent and selective nAChR antagonist was labeled and characterized with PET in non-human primates. Its high brain uptake, high signal-to-noise ratio, and high specific binding strongly suggest a great potential to carry out imaging studies in humans. In addition, the use of a C-11 radiotracer would allow us to perform multiple PET studies in the same individual within a short time frame. The presence of an iodine atom in the molecule also allows the possibility to label with radioiodine for SPECT studies.

Septal innervation regulates the function of alpha7 nicotinic receptors in CA1 hippocampal interneurons.

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.

Interaction of corticosterone and nicotine in regulation of prepulse inhibition in mice.

The aim of the present study was to investigate if different levels of circulating corticosterone (CORT) modulate the effect of nicotine on prepulse inhibition (PPI), a measure of sensorimotor gating that is disrupted in schizophrenia and other mental illnesses. Four groups of mice were investigated: sham-operated, adrenalectomized (ADX) and implanted with a cholesterol pellet, ADX and implanted with a 10 mg CORT pellet, or ADX and 50 mg of CORT. Different CORT levels or doses of nicotine did not significantly affect startle responses. Baseline PPI was significantly reduced in mice implanted with the highest dose of CORT. In ADX mice implanted with cholesterol, nicotine treatment influenced PPI depending on the prepulse intensity. In ADX mice implanted with 50 mg of CORT, treatment with 10 mg/kg of nicotine caused a significant increase in PPI at all prepulse intensities. Binding studies showed that corticosterone treatment had significantly affected nicotinic acetylcholine receptor (nAChR) density in the mouse brain. Treatment with 50 mg CORT decreased 125I-epibatidine binding in the globus pallidus and 125I-alpha-bungarotoxin binding in the claustrum. These results suggest a possible interaction of corticosterone and nicotine at the level of the alpha4- and alpha7-type nAChR in the regulation of PPI. In situations of high circulating levels of corticosterone, nicotine may be beneficial to restore disruption of PPI.

Future therapeutic strategies in autoimmune myasthenia gravis.

Antibodies against muscle acetylcholine receptor (AChR) undoubtedly play a critical role in the pathology of most myasthenia gravis (MG) cases. Selective elimination of the majority of these antibodies should result in a considerable improvement of the MG symptoms. Such a specific elimination could be achieved by AChR-based immunoadsorbents. However, sufficient quantities of native human AChR are not available while bacterially expressed recombinant domains of the AChR are unable to bind satisfactorily MG antibodies. We have undertaken the production of the extracellular domains of human AChR subunits in eukaryotic systems, in native-like conformation, for their use as potent immunoadsorbents. The N-terminal extracellular domain (amino acids 1-210; alpha(1-210)) of the alpha(1) subunit of the human muscle AChR was expressed in the yeast Pichia pastoris. The polypeptide was water-soluble, glycosylated, and in monomer form. The alpha(1-210) bound 125I-alpha-bungarotoxin (125I-alpha-BTX) with a high affinity (Kd = 5.1 +/- 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine. Several conformation-dependent anti-AChR antibodies were able to bind alpha(1-210) as did antibodies from a large proportion of MG patients. The purified protein was subsequently immobilized on Sepharose-CNBr and was used to immunoadsorb anti-AChR antibodies from 64 MG sera. It eliminated more than 50% (50-94%) of the anti-AChR antibodies in 20% of the sera, whereas from another 30% of the sera it eliminated 20-60% of their anti-AChR antibodies. Work is in progress for the expression of the extracellular domain of all other muscle AChR subunits. It is expected that their combined use may eliminate the great majority of the anti-AChR antibodies from most MG patients.

5-Iodo-A-85380 binds to alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) as well as alpha4beta2* subtypes.

Recent work suggests that 5-iodo-A-85380, a radioiodinated analog of the 3-pyridyl ether A-85380, represents a promising imaging agent for non-invasive, in vivo studies of alphaAbeta2* nicotinic acetylcholine receptors (nAChRs; *denotes receptors containing the indicated subunits), because of its low non-specific binding, low in vivo toxicity and high selectivity for alpha4beta2* nAChRs. As an approach to elucidate nAChR subtypes expressed in striatum, we carried out competitive autoradiography in monkey and rat brain using 5-[125I]iodo-A-85380 ([125I]A-85380) and [125I]alpha-conotoxin MII, a ligand that binds with high affinity to alpha6* and alpha3* nAChRs, but not to alpha4beta2* nAChRs. Although A-85380 is reported to be selective for alpha4beta2* nAChRs, we observed that A-85380 completely inhibited [125I]alpha-conotoxin MII binding in rat striatum and that A-85380 blocked >90% of [125I] alpha-conotoxin MII sites in monkey caudate and putamen. These results suggest that A-85380 binds to non-alpha4beta2* nAChRs, including putative alpha6* nAChRs. Experiments to determine the percentage of [125I]A-85380 sites that contain alpha-conotoxin MII-sensitive (alpha6beta2*) nAChRs indicate that they represent about 10% of [125I]A-85380 sites in rodent striatum and about 30% of sites in monkey caudate and putamen. These data are important for identifying alterations in nicotinic receptor subtypes in Parkinson's disease and other basal ganglia disorders both in in vitro and in in vivo imaging studies.

Simultaneous determination of mecamylamine, nicotine, and cotinine in plasma by gas chromatography-mass spectrometry.

The nicotine receptor antagonist mecamylamine has been shown to increase the efficacy of transdermal nicotine as a pharmacotherapy for tobacco addiction. A product for simultaneous transdermal administration of nicotine and mecamylamine is undergoing clinical trials. In order to carry out pharmacokinetic studies, quantitation of low nanogram per milliliter levels of mecamylamine and nicotine was required. This paper describes a method for simultaneous determination of mecamylamine, nicotine, and the nicotine metabolite, cotinine, in human plasma using gas chromatography-mass spectrometry (GC-MS). Limits of quantitation for mecamylamine, nicotine and cotinine are 2, 1 and 2 ng/ml, respectively.

Persistent nicotinic blockade by chlorisondamine of noradrenergic neurons in rat brain and cultured PC12 cells.

Chlorisondamine (CHL) blocks behavioural responses to nicotine for several weeks or months in rats. Persistent blockade has also been demonstrated ex vivo, in assays of nicotine-evoked striatal dopamine release. Central administration of [3H]-CHL leads to long-term retention of radiolabel in nigrostriatal dopaminergic neurons and in few other cell groups. We investigated whether an analogous blockade also occurs in noradrenergic neurons in the brain and in cultured pheochromocytoma (PC12) cells, which have a similar noradrenergic phenotype. Administration of CHL (10 mg kg(-1) s.c. or 10 microg i.c.v.), 21 days prior, resulted in a near-total block of nicotine-evoked release of hippocampal [3H]-noradrenaline ([3H]-NA) from superfused rat synaptosomes; NMDA-evoked [3H]-NA release was unaffected. Three weeks after administration of [3H]-CHL (10 microg i.c.v.), preferential accumulation of radiolabel was observed in the locus coeruleus, which provides the entire noradrenergic innervation to hippocampus, as well as in previously noted structures. In rat pheochromocytoma (PC12) cells, nicotine evoked [3H]-NA release (EC50 approximately 30 microM). This effect was blocked by co-incubation with mecamylamine (10 microM) or CHL (1 microM) but was not affected by alpha-bungarotoxin. As in the hippocampus, the nicotinic agonist cytisine was at least as efficacious as nicotine. Acute exposure of PC12 cells to CHL 10 or 100 microM (but not 1 microM), followed by 90 min wash-out, almost completely blocked release evoked by 30 microM nicotine. More prolonged (24 h) exposure to CHL 100 microM (but not 1 or 10 microM), followed by 3 days of wash-out, partially inhibited release evoked by nicotine, leaving responses to high K+ unchanged. A significant (30%) reduction was also seen 5 days after exposure. We conclude that persistent nicotinic blockade by CHL is neither restricted to mesostriatal dopamine neurons, nor to the CNS, nor to neurons possessing the same nicotinic receptor pharmacology. In addition, the persistent blockade does not appear to result from an acute blocking action, but may be dependent upon intracellular accumulation of the antagonist.

Intraneuronal accumulation and persistence of radiolabel in rat brain following in vivo administration of [3H]-chlorisondamine.

1. Chlorisondamine (CHL), a bisquaternary amine, produces a remarkably long-lasting blockade of central responses to nicotine. The mechanism underlying this blockade is not known. The main aim of this study was to test for possible accumulation of [3H]-CHL in rat brain during the period of chronic blockade. 2. Rats received CHL, either systemically (10 mg kg-1) or centrally (10 micrograms i.c.v.). Seven days later, striatal synaptosomes prepared from these animals were tested for nicotine-induced [3H]-dopamine release. This experiment showed that i.c.v. administration of CHL was as effective as systemic administration in producing ex vivo blockade of central nicotinic receptors. 3. Rats received bilateral i.c.v. infusions of [3H]-CHL (10 micrograms) and radioactivity was subsequently quantified in dissected cerebral cortex, striatum, hippocampus, midbrain and cerebellum. Radiolabel was detected at all three survival times (1, 7, and 21 days). Regional heterogeneity was apparent at 7 and 21 days survival. Radiolabel was almost exclusively confined to the insoluble subcellular fraction in all areas sampled. 4. The anatomical distribution of radiolabel was also visualized in brain sections. Rats received bilateral i.c.v. infusions of [3H]-CHL (10 micrograms) and were killed at 1, 7, 21 or 84 days. Immediately before they were killed, all rats were tested behaviourally, and central nicotinic blockade was demonstrated at 1, 7 and 21 days; partial recovery was observed at 84 days. Particularly at longer survival times, tritium was found to be heavily concentrated in the substantia nigra pars compacta, ventral tegmental area, dorsal raphé nucleus, and the granular layer of the cerebellum. 5. The possibility of retrograde axonal transport of radiolabel was then examined. Rats received a unilateral intrastriatal infusion of [3H]-CHL (0.34 or 0.034 micrograms) one week before they were killed. Autoradiographic labelling was largely confined to the site of infusion and to the ipsilateral substantia nigra pars compacta and dorsal raphé nucleus. 6. Thus, after i.c.v. administration, CHL (and/or centrally-formed derivatives) is initially widely distributed within the brain and is then selectively retained within a few brain areas. A persistent accumulation occurs within putative dopaminergic and 5-hydroxytryptaminergic neurones, at least partly through uptake by terminals and/or axons followed by retrograde transport. This persistent and anatomically-selective intraneuronal accumulation possibly underlies the long-term central nicotinic blockade associated with chlorisondamine.