Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades α e β. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo α3β4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade α3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR α3β4 de rato recombinante expresso nas células KXα3β4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 µM e a constante de equilíbrio de abertura do canal, Φ-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 µM. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by α and β subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The α3β4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; α3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat α3β4 nAChR expressed in KXα3β4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 µM and the channel-opening equilibrium constant, Φ-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 µM.

Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.

Objective: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. Methods: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. Results: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. Conclusions: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

Recently, the medial habenula-interpeduncular (MHb-IPN) axis has been hypothesized to modulate anxiety although neuronal populations and molecular mechanisms regulating affective behaviors in this circuit are unknown. Here we show that MHb cholinergic neuron activity directly regulates anxiety-like behavior. Optogenetic silencing of MHb cholinergic IPN inputs reduced anxiety-like behavior in mice. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their role as autoreceptors in these neurons has not been described. nAChRs are ligand-gated cation channels that are activated by the excitatory neurotransmitter, acetylcholine (ACh), as well as nicotine, the addictive component of tobacco smoke. We expressed novel nAChR subunits that render nAChRs hypersensitive to ACh, ACh detectors, selectively in MHb cholinergic neurons of adult mice. Mice expressing these ACh detectors exhibited increased baseline anxiety-like behavior that was alleviated by blocking the mutant receptors. Under stressful conditions, such as during nicotine withdrawal, nAChRs were functionally upregulated in MHb cholinergic neurons mediating an increase in anxiety-like behavior. Together, these data indicate that MHb cholinergic neurons regulate anxiety via signaling through nicotinic autoreceptors and point toward nAChRs in MHb as molecular targets for novel anxiolytic therapeutics.

Alcohol, recently named the most dangerous drug in the world, contributes to nearly 40% of violent crimes and fatal traffic accidents, increases risk of roughly 60 different diseases and injuries, and is responsible for 2.5 million deaths each year worldwide. Despite these staggering figures, treatments remain ineffective and riddled with adverse side effects, making successful use of even the most effective treatments unlikely. Moreover, many of the treatments, and the supporting research, have focused only on male subjects, despite sex differences in various alcohol-related behaviors. Human alcohol use is frequently accompanied by nicotine use, and vice versa, suggesting a common mechanism of the two drugs. In fact, alcohol may act through the same family of receptors as nicotine, the nicotinic acetylcholine receptors (nAChRs), eliciting similar activation of the reward pathway as nicotine and other drugs of abuse. Studies have shown that nAChRs containing the α4 and/or α6 subunits are involved in nicotine-induced activation of the reward pathway, leading to the hypothesis that these same receptor subtypes may be important for alcohol effects in the brain as well. Using male and female genetic mouse models and various behavioral assays, we have shown not only that these α4 and/or α6-containing nAChRs are involved in alcohol- related behaviors and activation of the reward pathway, but also show sex differences in this involvement. Uncovering the mechanism of alcohol in the brain, in males as well as in females, is an important step in developing targeted treatments for alcohol abuse.

No description available.

No description available.

The subcellular mechanism and site of action of linopirdine or DuP 996 (3,3‐bis(4‐pyridinylmethyl)‐1‐phenylindolin‐2‐one) was investigated at the frog neuromuscular junction, using miniature endplate potential (m.e.p.p.) counts and a new method for obtaining unbiased estimates of n (number of functional release sites), p (probability of release), and varsp (spatial variance in p). DuP 996 produced an increase in m (no. of quanta released), which was due to an increase in n and p. The increase in m was concentration‐dependent over a range of 0.1–100 μm and completely reversible with 15 min of wash. There was a saturation in the increase in p, but not in the increase in m and n, for [DuP 996] >10 μm. By contrast, there was no major change in varsp. Block of presynaptic Na+‐ and Ca2+‐channels with 3 μm tetrodotoxin and 1.8 mm Co2+prevented the m.e.p.p. frequency increase to DuP 996, and this effect was completely reversed by washing. Application of the neuronal Ca2+‐channel blocker, ω‐conotoxin GVIA (1 μm) brought about a rapid and profound decrease in the m.e.p.p. frequency increase produced by DuP 996. The effect of the toxin was not reversed by prolonged washing. Block of voltage‐gated K+‐channels with 100 μm 4‐aminopyridine (4‐AP) resulted in only a small (28%) increase in m. The combination of 4‐AP (100 μm) and DuP 996 (10 μm) produced an increase in m (189%) which was much greater than the sum of the responses to each agent alone. This increase in m was due solely to an increase in n, as p and varsp were unchanged. For [DuP 996] up to 100 μm, there was no apparent change in the mean size, amplitude distribution, or time course of m.e.p.ps, signifying that it had no anticholinesterase activity. It is concluded that DuP 996 increases the release of quantal transmitter but not the postsynaptic response to the quanta. This appears to involve an effect at the nerve terminal membrane, most likely an increase in Ca2+‐conductance, and not an action to block K+‐conductance or to release Ca2+from intraterminal organelles. 1994 British Pharmacological Society

Indiana University-Purdue University Indianapolis (IUPUI) / Nicotine use among individuals with schizophrenia occurs at extremely high rates. The prevailing theory is that individuals with schizophrenia smoke as a form of self-medication to ameliorate sensory and cognitive deficits. However, these individuals also have enhanced rates of addiction to several drugs of abuse and may therefore smoke as a result of enhanced addiction liability. The experiments described herein explored these two hypotheses by assessing the effect that nicotine has on working memory, addiction vulnerability (locomotor sensitization and self-administration), and nicotinic acetylcholine receptor (nAChR) expression as well as the developmental expression of these characteristics in the neonatal ventral hippocampal (NVHL) neurodevelopmental animal model of schizophrenia. The results from these studies indicate that NVHLs had working memory impairments in both adolescence and adulthood, with nicotine having a negligible effect. Additionally, NVHLs displayed enhanced locomotor sensitization to nicotine which emerged in adulthood as well as an enhanced acquisition of nicotine self-administration, administering more nicotine overall. These behavioral differences cannot be attributed to nAChR expression as nicotine upregulated nAChR to a similar extent between NVHL and SHAM control animals. These data indicate that the enhanced rates of nicotine use among individuals with schizophrenia may occur as a result of an enhanced vulnerability to nicotine addiction.

Rats were administered nicotine (0.3 mg/kg) for 11 consecutive days before and after an electrolytic medial frontal cortex lesion. Behavioral testing was arranged so that the rats were tested on the RAM 1 day after drug administration followed by behavioral testing on the MWT 19 days after drug treatment, or tested on the MWT 1 day after drug administration followed by testing on the RAM4 days after drug treatment. Results of MWT testing showed that regardless of the drug/behavioral testing interval, lesioned rats given nicotine demonstrated enhancement relative to saline-treated animals. Results of RAM testing showed that nicotine improved performance in non-lesioned rats compared to non-lesioned rats given saline. Four days after drug administration, nicotine improved performance in lesioned rats to levels of non-lesioned rats regardless of drug treatment. A second experiment was implemented to determine if the previous training on the MWT affected performance on the RAM.