Receptores nicotínicos da acetilcolina são encontrados na retina de pintos desde o início do desenvolvimento embrionário. As propostas desse trabalho foram caracterizar esses receptores no desenvolvimento de células de retinas embrionárias de pinto com oito dias, em cultura, e investigar se luzindole, um antagonista de receptores de melatonina, interfere com a atividade, distribuição e número desses receptores. Os ensaios funcionais foram feitos através de microfisiometria, método no qual é medido o aumento da velocidade de acidificação do meio extracelular de células em cultura, provocado pela ativação de receptores por agonistas. Os resultados são expressos como o percentual de aumento da velocidade de acidificação do meio extracelular acetilcolina-estimulado sobre a velocidade de acidificação do meio extracelular basal (ECAR % basal). A eficácia da acetilcolina aumentou do quarto dia de cultura para o quinto dia, decaindo ao oitavo dia, sendo bloqueada de modo dependente de concentração por dihidro-β-eritroidina (a partir de 10 µM), ao quarto dia e por α-bungarotoxina (10nM), ao quinto e sexto dia de cultivo, não ocorrendo o inverso. Para os ensaios de ligação, utilizou-se [125I] α-bungarotoxina, e ao quarto dia de cultivo houve maior número de sítios, menor afinidade e maior grau de cooperatividade. Ao quinto dia de cultivo ocorreu o inverso. Foi investigado, por imunocitoquímica, o desenvolvimento da distribuição da imunorreatividade para as subunidades α3 e α8 e ambas foram encontradas em culturas de quatro e seis dias, estando α3 principalmente em corpos neuronais e dendritos proximais e α8 principalmente em prolongamentos. O tratamento crônico com luzindole não interferiu com o padrão de distribuição das subunidades α3 e α8 em culturas de quatro ou seis dias, em nenhum tempo de cultivo. Também não interferiu no número de sítios, na constante de associação e no tempo de equilíbrio da ligação de [125I] α-bungarotoxina, nas culturas cultivadas por cinco dias. Entretanto, a resposta à acetilcolina em culturas de cinco e seis dias foi inibida de modo concentração e tempo dependente por luzindole, sem apresentar somação com a inibição por α-bungarotoxina. Concluiu-se que na cultura de células de retina a eficácia do agonista acetilcolina é dependente do desenvolvimento, se deve principalmente a receptores formados de subunidades α3 e α8, ao quarto e quinto dias de cultivo, respectivamente, e que o bloqueio de receptores de melatonina com luzindole inibe a resposta à acetilcolina somente ao quinto e sexto dias de desenvolvimento, provavelmente, pela inibição de outro sistema de neurotransmissão, localizado nos mesmos neurônios que contém receptores sensíveis a α-bungarotoxina em suas ramificações, como o sistema glutamatérgico. / Nicotinic acetylcholine receptors are expressed in the chick retina very early in embryonic development. The present study aimed to characterize the nicotinic acetylcholine receptors during embryonic chick retinal cell culture development and to investigate if luzindole, a melatonin receptor antagonist, is able to change the activity, distribution, and number of these receptors. The functional assays were done by microphysiometry, a method in which the increasing, agonist-stimulated, extracellular acidification rate is measured in cultured cells. The results are expressed in terms of the percentual of the acetylcholine-stimulated extracellular acidification rate over the basal extracellular acidification rate (% basal ECAR). The acetylcholine efficacy increased from the fourth to the fifth day, diminishing at the eighth culture day, and was inhibited, concentration-dependently, by dihydro-β-erythroidine (starting at 10 µM), in the retinal cells cultured for four days, and by α-bungarotoxin (10nM), in the retinal cells cultured for five and six days. The opposite did not occur. We have used [125I] α-bungarotoxin for the binding assays, and retinal cells cultured for four days presented in these assays a higher maximal-binding, smaller affinity, and higher degree of the cooperativity than retinal cells cultured for five days. Immunocytochemistry was used to characterize the development of the α3 and α8 subunits. Each of these subunits was characteristically distributed throughout the cell, independent of the age of culture. Alpha3 was mainly observed in the perikarya and proximal dendrites, whereas α8 was basically seen in processes. The distribution of the α3 and α8 immunoreactivity was not changed after chronic luzindole treatment. Also, the time of the equilibrium, the association rate, and the number of the [125I] α-bungarotoxin (10nM) binding sites were not different with or without chronic luzindole treatment in cells cultured for five days. However, the acetylcholine efficacy in the retinal cells cultured for five and six days was inhibited by luzindole, an effect that was concentration and time dependent, and that exhibited no summation with the inhibition by α-bungarotoxin. In conclusion, the acetylcholine efficacy is dependent on retinal cell culture development, and it acts mainly through neuronal nicotinic receptors comprising α3 subunits in the fourth day, and α8 subunits in the fifth day. Acetylcholine action is inhibited by melatonin receptor blockage by luzindole only at the fifth and sixth days, probably by inhibition of other receptors located in the same cells that harbor α-bungarotoxin-sensitive receptors, such as glutamate receptors.

Receptores nicotínicos da acetilcolina são encontrados na retina de pintos desde o início do desenvolvimento embrionário. As propostas desse trabalho foram caracterizar esses receptores no desenvolvimento de células de retinas embrionárias de pinto com oito dias, em cultura, e investigar se luzindole, um antagonista de receptores de melatonina, interfere com a atividade, distribuição e número desses receptores. Os ensaios funcionais foram feitos através de microfisiometria, método no qual é medido o aumento da velocidade de acidificação do meio extracelular de células em cultura, provocado pela ativação de receptores por agonistas. Os resultados são expressos como o percentual de aumento da velocidade de acidificação do meio extracelular acetilcolina-estimulado sobre a velocidade de acidificação do meio extracelular basal (ECAR % basal). A eficácia da acetilcolina aumentou do quarto dia de cultura para o quinto dia, decaindo ao oitavo dia, sendo bloqueada de modo dependente de concentração por dihidro-β-eritroidina (a partir de 10 µM), ao quarto dia e por α-bungarotoxina (10nM), ao quinto e sexto dia de cultivo, não ocorrendo o inverso. Para os ensaios de ligação, utilizou-se [125I] α-bungarotoxina, e ao quarto dia de cultivo houve maior número de sítios, menor afinidade e maior grau de cooperatividade. Ao quinto dia de cultivo ocorreu o inverso. Foi investigado, por imunocitoquímica, o desenvolvimento da distribuição da imunorreatividade para as subunidades α3 e α8 e ambas foram encontradas em culturas de quatro e seis dias, estando α3 principalmente em corpos neuronais e dendritos proximais e α8 principalmente em prolongamentos. O tratamento crônico com luzindole não interferiu com o padrão de distribuição das subunidades α3 e α8 em culturas de quatro ou seis dias, em nenhum tempo de cultivo. Também não interferiu no número de sítios, na constante de associação e no tempo de equilíbrio da ligação de [125I] α-bungarotoxina, nas culturas cultivadas por cinco dias. Entretanto, a resposta à acetilcolina em culturas de cinco e seis dias foi inibida de modo concentração e tempo dependente por luzindole, sem apresentar somação com a inibição por α-bungarotoxina. Concluiu-se que na cultura de células de retina a eficácia do agonista acetilcolina é dependente do desenvolvimento, se deve principalmente a receptores formados de subunidades α3 e α8, ao quarto e quinto dias de cultivo, respectivamente, e que o bloqueio de receptores de melatonina com luzindole inibe a resposta à acetilcolina somente ao quinto e sexto dias de desenvolvimento, provavelmente, pela inibição de outro sistema de neurotransmissão, localizado nos mesmos neurônios que contém receptores sensíveis a α-bungarotoxina em suas ramificações, como o sistema glutamatérgico. / Nicotinic acetylcholine receptors are expressed in the chick retina very early in embryonic development. The present study aimed to characterize the nicotinic acetylcholine receptors during embryonic chick retinal cell culture development and to investigate if luzindole, a melatonin receptor antagonist, is able to change the activity, distribution, and number of these receptors. The functional assays were done by microphysiometry, a method in which the increasing, agonist-stimulated, extracellular acidification rate is measured in cultured cells. The results are expressed in terms of the percentual of the acetylcholine-stimulated extracellular acidification rate over the basal extracellular acidification rate (% basal ECAR). The acetylcholine efficacy increased from the fourth to the fifth day, diminishing at the eighth culture day, and was inhibited, concentration-dependently, by dihydro-β-erythroidine (starting at 10 µM), in the retinal cells cultured for four days, and by α-bungarotoxin (10nM), in the retinal cells cultured for five and six days. The opposite did not occur. We have used [125I] α-bungarotoxin for the binding assays, and retinal cells cultured for four days presented in these assays a higher maximal-binding, smaller affinity, and higher degree of the cooperativity than retinal cells cultured for five days. Immunocytochemistry was used to characterize the development of the α3 and α8 subunits. Each of these subunits was characteristically distributed throughout the cell, independent of the age of culture. Alpha3 was mainly observed in the perikarya and proximal dendrites, whereas α8 was basically seen in processes. The distribution of the α3 and α8 immunoreactivity was not changed after chronic luzindole treatment. Also, the time of the equilibrium, the association rate, and the number of the [125I] α-bungarotoxin (10nM) binding sites were not different with or without chronic luzindole treatment in cells cultured for five days. However, the acetylcholine efficacy in the retinal cells cultured for five and six days was inhibited by luzindole, an effect that was concentration and time dependent, and that exhibited no summation with the inhibition by α-bungarotoxin. In conclusion, the acetylcholine efficacy is dependent on retinal cell culture development, and it acts mainly through neuronal nicotinic receptors comprising α3 subunits in the fourth day, and α8 subunits in the fifth day. Acetylcholine action is inhibited by melatonin receptor blockage by luzindole only at the fifth and sixth days, probably by inhibition of other receptors located in the same cells that harbor α-bungarotoxin-sensitive receptors, such as glutamate receptors.

Abstract not available

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 153-165).

This project utilized synthesis and in vitro assays to generate antagonist SARs at various nAChR subtypes. Alkylation of the pyridino nitrogen of the nicotine molecule afforded subtype specific antagonists at a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Using this data, a series of mono-azaaromatic quaternary salts were produced and evaluated in binding and functional assays for a42* and a7* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Additionally, bis-azaaromatic quaternary salts were synthesized and evaluated in the same assays. Two potent lead compounds were identified. N-n-dodecylnicotinium iodide (NDDNI) was found to be very potent at both a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. And the most promising candidate was N-N-bisdodecylpicolinium dibromide (bDDPiB), which was selective for the nAChR subtypes that mediate nicotine-evoked dopamine release (IC50 = 9 nM). Additionally, using the data from the SARs, predictive computer models were generated to assist in future compound assessment without in vitro assays. Three self-organizing map (SOMs) models were generated from three different sets of compounds. The groups consisted of the mono-substituted compounds, the bissubstituted compounds, and both sets combined. The models were able to successfully "bin" the test set of compounds after developing a model from a similar set of training compounds. Additionally, using genetic functional activity (GFA) algorithms an evolutionary approach to generating predictive model equations was applied to the compounds. Three separate equations were generated in order to form a predictive method for evaluating affinities at the a4b2* receptor subtype. In addition to the modeling and SAR work of the quaternary ammonium compounds, novel synthetic methods were also employed to develop enantiomerically pure nicotine analogs. Efficient enantioselective syntheses of (S)- and R-(+)-nornicotine, (S)-and R-(+)-anabasine, and (S)-and R-(+)-anatabine have been developed, affording isomers in high enantiomeric excess.

The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Tobacco dependence is high in women who suffer from anxiety disorders yet little is known about the contributions of nicotinic acetylcholine receptors (nAChRs) on anxiety-like behavior. β2*nAChRs (*denotes assembly with other subunits) are the most abundantly expressed nAChRs in the brain yet little is known about the contributions of β2*nAChRs on anxiety-like behavior in female mice. In this study, antagonism and nicotine effects on anxiety-like behavior was investigated across the life span in 6, 12 and 24-month-old drug-naïve knockout (KO), heterozygous (HET) and a gain of function α6L9S mice and wild type (WT). HET mice showed increased sensitivity to di-hydrobeta-erythroidine compared to WT mice. Aged mice showed decreased locomotor activity and exploratory behavior compared to younger mice. Low doses of nicotine produced anxiolytic-like effects, whilst a high dose of nicotine produced anxiogenic-like effects. Activation of the α6*nAChRs supports an anxiolysis-like phenotype. These results implicate α4β2*nAChRs and α6β2*nAChRs in anxiety-like behavior.

Tobacco dependence is the leading cause of preventable death in the United States. Despite currently available smoking cessation therapies, there is a high rate of relapse in smoking among those attempting to quit. While the somatic signs of nicotine withdrawal (insomnia, increased appetite, weight gain) contribute to the continuation of smoking behavior, it has been hypothesized that the affective signs (depression, anxiety, craving, irritability) are greater motivators of relapse and continued tobacco use. There are few studies that assess the molecular and receptor-mediated mechanisms of nicotine withdrawal; therefore, our studies focus on identifying the nicotinic acetylcholine receptor (nAChR) subtypes and post-receptor calcium-dependent mechanisms involved in nicotine withdrawal behaviors. Using precipitated, spontaneous, and conditioned place aversion (CPA) models, we measured physical and affective signs of nicotine withdrawal in mice. Our data show that major nAChR subtypes have differential roles in nicotine withdrawal. Additionally, our results suggest a behavioral relevance for L-type calcium channels in physical nicotine withdrawal signs, while calcium/calmodulin dependent protein kinase II (CaMKII) appears to be involved in both physical and affective withdrawal behaviors. Additionally, we conducted biochemical studies in the ventral tegmental area (VTA) and nucleus accumbens (NAc) to examine the relationship between altered withdrawal behavioral responses and calcium-dependent molecular mechanisms that contribute to nicotine withdrawal behaviors. Our results suggest an important role for β2-containing nAChRs in nicotine-withdrawal induced decreases in CaMKII and synapsin I function in the NAc. Overall, our studies implicate a critical role for the α4α6β2* nAChR subtype in the behavioral and molecular aspects of nicotine withdrawal, thus aiding in the elucidation of nAChR subunits and mechanisms that contribute to nicotine withdrawal behaviors. The current studies are imperative for generating more successful smoking cessation therapies.

Neuronal nicotinic acetylcholine receptors modulate both cholinergic and non-cholinergic synaptic transmission. Our research concerns α6 and α4 neuronal nicotinic subunits because they often co-assemble with the β2 subunit, which has abundant expression in the CNS and previous work has demonstrated that β2* nAChRs are involved in nicotine and cocaine reward. α6β2* and α4β2* nAChRs are highly expressed in midbrain, which is known to be critical for the incentive salience associated with natural and artificial (drug) reward. Our goal was to assess the role of α6β2* and α4β2* nAChRs in nicotine and cocaine reward using an unbiased conditioned place preference (CPP) test in mice. Adult male C57BL/6J mice or male mice null for the α6 or α4 nicotinic receptor subunit were used. For CPP: On day 1, pre-conditioning scores were recorded; Days 2-4 mice underwent conditioning, where they were randomly assigned to either the black or the white compartment paired with drug, and the opposite chamber paired with saline; Day 5 was a drug-free test day where post conditioning scores were recorded. α-Conotoxin MII[H9A;L15A], a selective antagonist of α6β2* nAChRs, was given centrally either into the lateral ventricle or the nucleus accumbens on conditioning days, which tested for acquisition of CPP, or it was given only once into the lateral ventricle on test day which tested for expression of CPP. Antagonizing α6*nAChRs resulted in a significant attenuation of both nicotine and cocaine place preference. This was complemented with diminished nicotine and cocaine place preference in α6 KO mice compared to WT littermates. Studies with α4 KO mice showed significantly reduced nicotine place preference scores compared to WT littermates. In contrast, α4 KO and WT mice showed significant place preference for 20mg /kg cocaine, suggesting that the α4 subunit is not required for the reward-like effects of cocaine in our behavioral test. Our results implicate α6β2* and α4β2* nAChR involvement in nicotine and cocaine CPP, but only α6β2* nAChR involvement in cocaine CPP. Lithium conditioned place avoidance and food reward were not altered in α6 KO mice or by α-Conotoxin MII[H9A;L15A], thereby validating the specificity of hedonics of targeting α6* nAChRs in CPP. Our studies suggest that α6β2* and α4β2*nAChR should be further characterized for future nicotine cessation therapies, and α6β2* could provide a new target for treating cocaine addiction.