RESUMO
Light-controllable tools provide powerful means to manipulate and interrogate brain function with relatively low invasiveness and high spatiotemporal precision. Although optogenetic approaches permit neuronal excitation or inhibition at the network level, other technologies, such as optopharmacology (also known as photopharmacology) have emerged that provide molecular-level control by endowing light sensitivity to endogenous biomolecules. In this Review, we discuss the challenges and opportunities of photocontrolling native neuronal signalling pathways, focusing on ion channels and neurotransmitter receptors. We describe existing strategies for rendering receptors and channels light sensitive and provide an overview of the neuroscientific insights gained from such approaches. At the crossroads of chemistry, protein engineering and neuroscience, optopharmacology offers great potential for understanding the molecular basis of brain function and behaviour, with promises for future therapeutics.
Assuntos
Canais Iônicos/metabolismo , Neurônios/metabolismo , Optogenética/tendências , Processos Fotoquímicos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Moduladores de Transporte de Membrana/farmacologia , Neurônios/química , Neurônios/efeitos dos fármacos , Optogenética/métodos , Processos Fotoquímicos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/químicaRESUMO
Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors at concentrations that are effective for photolysis with violet light. Pharmacological tests in vitro revealed that attachment of a fifth-generation (G5) dendrimer (i.e., cloaking) to the widely used 4-methoxy-7-nitro-indolinyl(MNI)-Glu probe prevented such off-target effects while not changing the photochemical properties of MNI-Glu significantly. G5-MNI-Glu was used with optofluidic delivery to stimulate dopamine neurons of the ventral tegmental area of freely moving mice in a conditioned place-preference protocol so as to mediate Pavlovian conditioning.
Assuntos
Glutamatos/farmacologia , Indóis/farmacologia , Aprendizagem/fisiologia , Microfluídica , Neurônios/fisiologia , Neurotransmissores/farmacologia , Animais , Aprendizagem/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neuroquímica , Neurônios/efeitos dos fármacos , Fotoquímica , Fotólise , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.
Assuntos
Canais Iônicos/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptividade/efeitos da radiação , Animais , Compostos Azo/química , Compostos Azo/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Canais Iônicos/antagonistas & inibidores , Camundongos , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/efeitos da radiação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Estimulação Luminosa , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Ratos , Receptores Purinérgicos P2X7/metabolismo , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/efeitos da radiação , Canais de Cátion TRPV/metabolismo , Fatores de TempoRESUMO
Cholinergic striatal interneurons (ChIs) express the vesicular glutamate transporter 3 (VGLUT3) which allows them to regulate the striatal network with glutamate and acetylcholine (ACh). In addition, VGLUT3-dependent glutamate increases ACh vesicular stores through vesicular synergy. A missense polymorphism, VGLUT3-p.T8I, was identified in patients with substance use disorders (SUDs) and eating disorders (EDs). A mouse line was generated to understand the neurochemical and behavioral impact of the p.T8I variant. In VGLUT3T8I/T8I male mice, glutamate signaling was unchanged but vesicular synergy and ACh release were blunted. Mutant male mice exhibited a reduced DA release in the dorsomedial striatum but not in the dorsolateral striatum, facilitating habit formation and exacerbating maladaptive use of drug or food. Increasing ACh tone with donepezil reversed the self-starvation phenotype observed in VGLUT3T8I/T8I male mice. Our study suggests that unbalanced dopaminergic transmission in the dorsal striatum could be a common mechanism between SUDs and EDs.
Assuntos
Corpo Estriado , Dopamina , Animais , Masculino , Dopamina/metabolismo , Camundongos , Corpo Estriado/metabolismo , Humanos , Acetilcolina/metabolismo , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Transtornos Relacionados ao Uso de Substâncias/genética , Transdução de Sinais/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Interneurônios/efeitos dos fármacos , Transtornos da Alimentação e da Ingestão de Alimentos/metabolismo , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Transtornos da Alimentação e da Ingestão de Alimentos/fisiopatologia , Camundongos Endogâmicos C57BL , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Mutação , Mutação de Sentido Incorreto , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Nicotine intake is likely to result from a balance between the rewarding and aversive properties of the drug, yet the individual differences in neural activity that control aversion to nicotine and their adaptation during the addiction process remain largely unknown. Using a two-bottle choice experiment, we observed considerable heterogeneity in nicotine-drinking profiles in isogenic adult male mice, with about half of the mice persisting in nicotine consumption even at high concentrations, whereas the other half stopped consuming. We found that nicotine intake was negatively correlated with nicotine-evoked currents in the interpeduncular nucleus (IPN), and that prolonged exposure to nicotine, by weakening this response, decreased aversion to the drug, and hence boosted consumption. Lastly, using knock-out mice and local gene re-expression, we identified ß4-containing nicotinic acetylcholine receptors of IPN neurons as molecular and cellular correlates of nicotine aversion. Collectively, our results identify the IPN as a substrate for individual variabilities and adaptations in nicotine consumption.
Assuntos
Habenula , Núcleo Interpeduncular , Receptores Nicotínicos , Camundongos , Masculino , Animais , Nicotina/farmacologia , Núcleo Interpeduncular/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Habenula/metabolismoRESUMO
The neural mechanisms by which animals initiate goal-directed actions, choose between options, or explore opportunities remain unknown. Here, we develop a spatial gambling task in which mice, to obtain intracranial self-stimulation rewards, self-determine the initiation, direction, vigor, and pace of their actions based on their knowledge of the outcomes. Using electrophysiological recordings, pharmacology, and optogenetics, we identify a sequence of oscillations and firings in the ventral tegmental area (VTA), orbitofrontal cortex (OFC), and prefrontal cortex (PFC) that co-encodes and co-determines self-initiation and choices. This sequence appeared with learning as an uncued realignment of spontaneous dynamics. Interactions between the structures varied with the reward context, particularly the uncertainty associated with the different options. We suggest that self-generated choices arise from a distributed circuit based on an OFC-VTA core determining whether to wait for or initiate actions, while the PFC is specifically engaged by reward uncertainty in action selection and pace.
Assuntos
Jogo de Azar , Camundongos , Animais , Aprendizagem/fisiologia , Dopamina , Córtex Pré-Frontal/fisiologia , Motivação , Área Tegmentar Ventral/fisiologia , RecompensaRESUMO
Long-term exposure to nicotine alters brain circuits and induces profound changes in decision-making strategies, affecting behaviors both related and unrelated to drug seeking and consumption. Using an intracranial self-stimulation reward-based foraging task, we investigated in mice the impact of chronic nicotine on midbrain dopamine neuron activity and its consequence on the trade-off between exploitation and exploration. Model-based and archetypal analysis revealed substantial inter-individual variability in decision-making strategies, with mice passively exposed to nicotine shifting toward a more exploitative profile compared to non-exposed animals. We then mimicked the effect of chronic nicotine on the tonic activity of dopamine neurons using optogenetics, and found that photo-stimulated mice adopted a behavioral phenotype similar to that of mice exposed to chronic nicotine. Our results reveal a key role of tonic midbrain dopamine in the exploration/exploitation trade-off and highlight a potential mechanism by which nicotine affects the exploration/exploitation balance and decision-making.
Assuntos
Comportamento Exploratório/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Nicotina/efeitos adversos , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Comportamento Exploratório/fisiologia , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Modelos Animais , Nicotina/administração & dosagem , Optogenética , Preconceito , Recompensa , Autoadministração , Técnicas EstereotáxicasRESUMO
Nicotine stimulates dopamine (DA) neurons of the ventral tegmental area (VTA) to establish and maintain reinforcement. Nicotine also induces anxiety through an as yet unknown circuitry. We found that nicotine injection drives opposite functional responses of two distinct populations of VTA DA neurons with anatomically segregated projections: it activates neurons that project to the nucleus accumbens (NAc), whereas it inhibits neurons that project to the amygdala nuclei (Amg). We further show that nicotine mediates anxiety-like behavior by acting on ß2-subunit-containing nicotinic acetylcholine receptors of the VTA. Finally, using optogenetics, we bidirectionally manipulate the VTA-NAc and VTA-Amg pathways to dissociate their contributions to anxiety-like behavior. We show that inhibition of VTA-Amg DA neurons mediates anxiety-like behavior, while their activation prevents the anxiogenic effects of nicotine. These distinct subpopulations of VTA DA neurons with opposite responses to nicotine may differentially drive the anxiogenic and the reinforcing effects of nicotine.
Assuntos
Ansiedade/tratamento farmacológico , Vias Neurais/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Ansiedade/induzido quimicamente , Ansiedade/fisiopatologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/fisiologia , Masculino , Camundongos , Vias Neurais/fisiologia , Nicotina/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Reforço Psicológico , Área Tegmentar Ventral/fisiologiaRESUMO
Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet they don't bind glutamate and are considered orphan. Progress in defining the ion channel function of GluDs in neurons has been hindered by a lack of pharmacological tools. Here, we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable pore blocker. In the constitutively open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with high pharmacological specificity that metabotropic glutamate receptor signaling triggers opening of GluD2. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.
Neurotransmitters are chemicals released by the body that trigger activity in neurons. Receptors on the surface of neurons detect these neurotransmitters, providing a link between the inside and the outside of the cell. Glutamate is one of the major neurotransmitters and is involved in virtually all brain functions. Glutamate binds to two different types of receptors in neurons. Ionotropic receptors have pores known as ion channels, which open when glutamate binds. This is a fast-acting response that allows sodium ions to flow into the neuron, triggering an electrical signal. Metabotropic receptors, on the other hand, trigger a series of events inside the cell that lead to a response. Metabotropic receptors take more time than ionotropic receptors to elicit a response in the cell, but their effects last much longer. One type of receptor, known as the GluD family, is very similar to ionotropic glutamate receptors but does not directly respond to glutamate. Instead, the ion channel of GluD receptors opens after being activated by glutamate metabotropic receptors. GluD receptors are produced throughout the brain and play roles in synapse formation and activity, but the way they work remains unclear. An obstacle to understanding how GluD receptors work is the lack of molecules that can specifically block these receptors' ion channel activity. Lemoine et al. have developed a tool that enables control of the ion channel in GluD receptors using light. Human cells grown in the lab were genetically modified to produce a version of GluD2 (a member of the GluD family) with a light-sensitive molecule attached. In darkness or under green light, the light-sensitive molecule blocks the channel and prevents ions from passing through. Under violet light, the molecule twists, and ions can flow through the channel. With this control over the GluD2 ion channel activity, Lemoine et al. were able to validate previous research showing that the activation of metabotropic glutamate receptors can trigger GluD2 to open. The next step will be to test this approach in neurons. This will help researchers to understand what role GluD ion channels play in neuron to neuron communication.
Assuntos
Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Sítios de Ligação , Engenharia Genética , Glutamato Desidrogenase/química , Glutamatos/química , Glutamatos/metabolismo , Células HEK293 , Humanos , Luz , MutaçãoRESUMO
Can decisions be made solely by chance? Can variability be intrinsic to the decision-maker or is it inherited from environmental conditions? To investigate these questions, we designed a deterministic setting in which mice are rewarded for non-repetitive choice sequences, and modeled the experiment using reinforcement learning. We found that mice progressively increased their choice variability. Although an optimal strategy based on sequences learning was theoretically possible and would be more rewarding, animals used a pseudo-random selection which ensures high success rate. This was not the case if the animal is exposed to a uniform probabilistic reward delivery. We also show that mice were blind to changes in the temporal structure of reward delivery once they learned to choose at random. Overall, our results demonstrate that a decision-making process can self-generate variability and randomness, even when the rules governing reward delivery are neither stochastic nor volatile.
Assuntos
Comportamento Animal , Comportamento de Escolha , Algoritmos , Animais , Teorema de Bayes , Aprendizagem , Masculino , Cadeias de Markov , Memória , Camundongos , Modelos TeóricosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Application of solid supported membranes (SSMs) for the functional investigation of ion channels is presented. SSM-based electrophysiology, which has been introduced previously for the investigation of active transport systems, is expanded for the analysis of ion channels. Membranes or liposomes containing ion channels are adsorbed to an SSM and a concentration gradient of a permeant ion is applied. Transient currents representing ion channel transport activity are recorded via capacitive coupling. We demonstrate the application of the technique to liposomes reconstituted with the peptide cation channel gramicidin, vesicles from native tissue containing the nicotinic acetylcholine receptor, and membranes from a recombinant cell line expressing the ionotropic P2X2 receptor. It is shown that stable ion gradients, both inside as well as outside directed, can be applied and currents are recorded with an excellent signal/noise ratio. For the nicotinic acetylcholine receptor and the P2X2 receptor excellent assay quality factors of Z' = 0.55 and Z' = 0.67, respectively, are obtained. This technique opens up new possibilities in cases where conventional electrophysiology fails like the functional characterization of ion channels from intracellular compartments. It also allows for robust fully automatic assays for drug screening.
Assuntos
Eletrofisiologia/métodos , Canais Iônicos/química , Membranas/química , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Órgão Elétrico/efeitos dos fármacos , Órgão Elétrico/fisiologia , Gramicidina/química , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Lipossomos/química , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Membranas/efeitos dos fármacos , Membranas/metabolismo , Antagonistas Nicotínicos/farmacologia , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X2 , Sódio/metabolismo , TorpedoRESUMO
Neuronal communication involves a multitude of neurotransmitters and an outstanding diversity of receptors and ion channels. Linking the activity of cell surface receptors and ion channels in defined neural circuits to brain states and behaviors has been a key challenge in neuroscience, since cell targeting is not possible with traditional neuropharmacology. We review here recent technologies that enable the effect of drugs to be restricted to specific cell types, thereby allowing acute manipulation of the brain's own proteins with circuit specificity. We highlight the importance of developing cell-specific neuropharmacology strategies for decoding the nervous system with molecular and circuit precision, and for developing future therapeutics with reduced side effects.
Assuntos
Sistema Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Humanos , Neurofarmacologia/métodosRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal alpha4beta4 nAChR by specifically tethering a conformation-sensitive fluorescent dye on alphaG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression in Xenopus oocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine-6-maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro-beta-erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.
Assuntos
Acetilcolina/farmacologia , Di-Hidro-beta-Eritroidina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/fisiologia , Animais , Cisteína/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microinjeções/métodos , Biologia Molecular , Mutação/fisiologia , Oocistos , Técnicas de Patch-Clamp , Receptores Nicotínicos/genética , Rodaminas/metabolismo , XenopusRESUMO
We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.
Assuntos
Proteínas de Peixes/agonistas , Proteínas de Peixes/metabolismo , Corantes Fluorescentes/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Torpedo , Acetilcolina/síntese química , Acetilcolina/farmacologia , Animais , Cumarínicos/química , Cumarínicos/farmacologia , Fluorescência , Corantes Fluorescentes/síntese química , Cinética , Agonistas Nicotínicos/síntese química , Oócitos/efeitos dos fármacos , Ensaio Radioligante , Especificidade por SubstratoRESUMO
Title: Prix Nobel de chimie 2022 - La chimie click et la chimie bio-orthogonale. Abstract: Selon Sydney Brenner, lauréat du prix Nobel de physiologie ou médecine en 2002, « les progrès de la science dépendent de nouvelles techniques, de nouvelles découvertes et de nouvelles idées, probablement dans cet ordre ¼. Le prix Nobel de chimie 2022 a été décerné à Carolyn Ruth Bertozzi (université de Stanford, États-Unis), Morten Peter Meldal (université de Copenhague, Danemark), et Karl Barry Sharpless (institut de recherche Scripps, La Jolla, États-Unis) pour le développement de la chimie click et de la chimie bio-orthogonale. Ce prix Nobel récompense dans une large mesure un développement conceptuel dans les techniques de synthèse chimique et de marquages des cellules, mais également des nouvelles découvertes, notamment en cancérologie. Morten Meldal et Barry Sharpless (qui obtient maintenant son deuxième prix Nobel de chimie, après celui de 2001 pour ses travaux sur la catalyse chirale de réactions d'oxydation) ont développé la chimie click, qui permet d'assembler des briques moléculaires rapidement et efficacement. Carolyn Bertozzi, quant à elle, a porté la chimie click à un autre niveau, en permettant son utilisation biologique sur des cellules et même chez l'animal in vivo.
Assuntos
Química Click , Prêmio Nobel , Humanos , QuímicaRESUMO
BACKGROUND AND PURPOSE: The photo-isomerizable local anaesthetic, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), provides rapid, optical control over pain signalling without involving genetic modification. In darkness or in green light, trans-QAQ blocks voltage-gated K+ and Na+ channels and silences action potentials in pain-sensing neurons. Upon photo-isomerization to cis with near UV light, QAQ blockade is rapidly relieved, restoring neuronal activity. However, the molecular mechanism of cis and trans QAQ blockade is not known. Moreover, the absorption spectrum of QAQ requires UV light for photo-control, precluding use deep inside neural tissue. EXPERIMENTAL APPROACH: Electrophysiology and molecular modelling were used to characterize the binding of cis and trans QAQ to voltage-gated K+ channels and to develop quaternary ammonium-ethylamine-azobenzene-quaternary ammonium (QENAQ), a red-shifted QAQ derivative controlled with visible light. KEY RESULTS: trans QAQ was sixfold more potent than cis QAQ, in blocking current through Shaker K+ channels. Both isomers were use-dependent, open channel blockers, binding from the cytoplasmic side, but only trans QAQ block was slightly voltage dependent. QENAQ also blocked native K+ and Na+ channels preferentially in the trans state. QENAQ was photo-isomerized to cis with blue light and spontaneously reverted to trans within seconds in darkness, enabling rapid photo-control of action potentials in sensory neurons. CONCLUSIONS AND IMPLICATIONS: Light-switchable local anaesthetics provide a means to non-invasively photo-control pain signalling with high selectivity and fast kinetics. Understanding the mode of action of QAQ and related compounds will help to design of drugs with improved photo-pharmacological properties. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
Assuntos
Compostos Azo/farmacologia , Luz , Nociceptores/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Compostos de Amônio Quaternário/farmacologia , Animais , Compostos Azo/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Nociceptores/metabolismo , Processos Fotoquímicos , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Compostos de Amônio Quaternário/químicaRESUMO
Tobacco addiction is a chronic and relapsing disorder with an important genetic component that represents a major public health issue. Meta-analysis of large-scale human genome-wide association studies (GWASs) identified a frequent non-synonymous SNP in the gene coding for the α5 subunit of nicotinic acetylcholine receptors (α5SNP), which significantly increases the risk for tobacco dependence and delays smoking cessation. To dissect the neuronal mechanisms underlying the vulnerability to nicotine addiction in carriers of the α5SNP, we created rats expressing this polymorphism using zinc finger nuclease technology and evaluated their behavior under the intravenous nicotine-self-administration paradigm. The electrophysiological responses of their neurons to nicotine were also evaluated. α5SNP rats self-administered more nicotine at high doses and exhibited higher nicotine-induced reinstatement of nicotine seeking than wild-type rats. Higher reinstatement was associated with altered neuronal activity in several discrete areas that are interconnected, including in the interpeduncular nucleus (IPN), a GABAergic structure that strongly expresses α5-containing nicotinic receptors. The altered reactivity of IPN neurons of α5SNP rats to nicotine was confirmed electrophysiologically. In conclusion, the α5SNP polymorphism is a major risk factor for nicotine intake at high doses and for relapse to nicotine seeking in rats, a dual effect that reflects the human condition. Our results also suggest an important role for the IPN in the higher relapse to nicotine seeking observed in α5SNP rats.
Assuntos
Nicotina/administração & dosagem , Polimorfismo de Nucleotídeo Único , Receptores Nicotínicos/genética , Autoadministração , Tabagismo/genética , Animais , Sequência de Bases , Feminino , Humanos , Masculino , Ratos , Ratos Long-Evans , Ratos Transgênicos , Receptores Nicotínicos/metabolismo , Recidiva , Alinhamento de SequênciaRESUMO
Dopamine (DA) neurons of the ventral tegmental area (VTA) integrate cholinergic inputs to regulate key functions such as motivation and goal-directed behaviors. Yet the temporal dynamic range and mechanism of action of acetylcholine (ACh) on the modulation of VTA circuits and reward-related behaviors are not known. Here, we used a chemical-genetic approach for rapid and precise optical manipulation of nicotinic neurotransmission in VTA neurons in living mice. We provide direct evidence that the ACh tone fine-tunes the firing properties of VTA DA neurons through ß2-containing (ß2*) nicotinic ACh receptors (nAChRs). Furthermore, locally photo-antagonizing these receptors in the VTA was sufficient to reversibly switch nicotine reinforcement on and off. By enabling control of nicotinic transmission in targeted brain circuits, this technology will help unravel the various physiological functions of nAChRs and may assist in the design of novel therapies relevant to neuropsychiatric disorders.