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Cigarette smoking is one of the most serious health problems worldwide and people trying to stop smoking have high rates of relapse. Zebrafish (Danio rerio), by combining pharmacological and behavioral assays, is a promising animal model for rapidly screening new compounds to induce smoking cessation.This study aims to identify possible acetylcholine nicotinic receptors (nAChRs) involved in mediating nicotine (NIC)-induced conditioned place preference (CPP) in zebrafish and investigate the effect of the CC4 and CC26 cytisine derivatives in reducing NIC-induced CPP.CPP was evaluated using a two-compartment chamber, and the zebrafish were given CC4 (0.001-5 mg/kg), CC26 (0.001-1 mg/kg), cytisine (0.1-2.5 mg/kg), and varenicline (1-10 mg/kg) alone or with NIC (0.001 mg/kg). Swimming activity was evaluated using a square observational chamber. The affinity of the nicotinic ligands for native zebrafish brain nAChRs was evaluated by binding studies using [(3)H]-Epibatidine (Epi) and [(125)I]-αBungarotoxin (αBgtx) radioligands, and their subtype specificity was determined by means of electrophysiological assay of oocyte-expressed α4β2 and α7 subtypes.CC4 and CC26 induced CPP with an inverted U-shaped dose-response curve similar to that of NIC. However, when co-administered with NIC, they blocked its reinforcing or slightly aversive effect. Binding and electrophysiological studies showed that this effect was due to binding to high-affinity heteromeric but not α7-containing receptors.We have further characterized CC4 and identified a new compound (CC26) that may be active in inducing smoking cessation. Zebrafish is a very useful model for screening new compounds that can affect the rewarding properties of NIC.

Background and Purpose Many of the addictive and rewarding effects of nicotine are due to its actions on the neuronal nicotinic ACh receptor (nAChR) subtypes expressed in dopaminergic mesocorticolimbic cells. The partial agonists, cytisine and varenicline, are helpful smoking cessation aids. These drugs have a number of side effects that limit their usefulness. The aim of this study was to investigate the preclinical pharmacology of the cytisine dimer1,2-bisN-cytisinylethane (CC4). Experimental Approach The effects of CC4 on nAChRs were investigated using in vitro assays and animal behaviours. Key Results When electrophysiologically tested using heterologously expressed human subtypes, CC4 was less efficacious than cytisine on neuronal α4β2, α3β4, α7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through α4β2 and α6β2 nAChRs, CC4 is a partial agonist of nAChR-mediated striatal dopamine release and, when co-incubated with nicotine, prevented nicotine's maximal effect on this response. In addition, it had low affinity for, and was less efficacious than nicotine and cytisine on the α3β4 and α7-nAChR subtypes. Like cytisine and nicotine, CC4-induced conditioned place preference (CPP), and its self-administration shows an inverted-U dose–response curve. Pretreatment with non-reinforcing doses of CC4 significantly reduced nicotine-induced self-administration and CPP without affecting motor functions. Conclusion and Implications Our in vitro and in vivo findings reveal that CC4 selectively reduces behaviours associated with nicotine addiction consistent with the partial agonist selectivity of CC4 for β2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels present in the central and peripheral nervous systems, that are permeable to mono- and divalent cations. They share a common basic structure but their pharmacological and functional properties arise from the wide range of different subunit combinations making up distinctive subtypes. nAChRs are involved in many physiological functions in the central and peripheral nervous systems, and are the targets of the widely used drug of abuse nicotine. In addition to tobacco dependence, changes in their number and/or function are associated with neuropsychiatric disorders, ranging from epilepsy to dementia. Although some of the neural circuits involved in the acute and chronic effects of nicotine have been identified, much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions. We briefly review some recent findings concerning the structure and function of native nAChRs, focusing on the subtypes identified in the mesostriatal and habenulo-interpeduncular pathways, two systems involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the effect of chronic nicotine on the expression of native subtypes.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10–32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.

Calcitonin (CT) measurement is crucial to the early diagnosis and the follow-up of medullary thyroid cancer (MTC). If the evaluation of stimulated CT levels is required, a provocative test can be performed, being the high-dose Ca test recently reintroduced in clinical practice.Our objective was to identify gender-specific thresholds for MTC diagnosis in a large series of patients who underwent the Ca test.A total of 91 patients (49 females and 42 males) underwent the Ca test (calcium gluconate, 25 mg/kg) before thyroidectomy and both basal CT (bCT) and stimulated CT (sCT) were compared with histological results by receiver operating characteristic plot analyses. To evaluate possible side effects of Ca administration, cardiac function has been extensively studied.bCT levels were found to harbor the same accuracy as sCT in the preoperative diagnosis of MTC. The best Ca thresholds for the identification of MTC were26 and68 for bCT and79 and544 pg/mL for sCT in females and males, respectively. The high tolerability and safety of the Ca test was demonstrated and advice offered to be followed before and during the test.Gender-specific bCT and sCT cutoffs for the identification of C-cell hyperplasia and/or MTC have been defined. The bCT and sCT were found to have a similar accuracy, indicating that serum CT assays with improved functional sensitivity may likely decrease the relevance of the stimulation test in several conditions. Finally, systematic cardiac monitoring confirms the safety of the Ca test.

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal form of epilepsy characterized by seizures occurring during non-REM sleep. We have developed and characterized the first mouse model for ADNFLE type III carrying the V287L mutation of the beta 2 subunit of neuronal nicotinic receptor. Mice expressing mutant receptors show a spontaneous epileptic phenotype by electroencephalography with very frequent interictal spikes and seizures. Expression of the mutant beta 2 subunit is driven by a neuronal-specific tetracycline-controlled promoter, which allows planned silencing of transgene expression in a reversible fashion and tracking the involvement of mutant receptor in crucial phases of epileptogenesis. We found that restricted silencing during development is sufficient to prevent the occurrence of epileptic seizures in adulthood. Our data indicate that mutant nicotinic receptors are responsible for abnormal formation of neuronal circuits and/or long-lasting alteration of network assembly in the developing brain, thus leading to epilepsy.

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the α4 and β2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor α2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic α2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior. © 2006 by The American Society of Human Genetics. All rights reserved.