RESUMEN
Self-awareness is a pivotal component of conscious experience. It is correlated with a paralimbic network of medial prefrontal/anterior cingulate and medial parietal/posterior cingulate cortical "hubs" and associated regions. Electromagnetic and transmitter manipulation have demonstrated that the network is not an epiphenomenon but instrumental in generation of self-awareness. Thus, transcranial magnetic stimulation (TMS) targeting the hubs impedes different aspects of self-awareness with a latency of 160ms. The network is linked by â¼40Hz oscillations and regulated by dopamine. The oscillations are generated by rhythmic GABA-ergic inhibitory activity in interneurons with an extraordinarily high metabolic rate. The hubs are richly endowed with interneurons and therefore highly vulnerable to disturbed energy supply. Consequently, deficient paralimbic activity and self-awareness are characteristic features of many disorders with impaired oxygen homeostasis. Such disorders may therefore be treated unconventionally by targeting interneuron function.
Asunto(s)
Concienciación/fisiología , Cognición/fisiología , Neurociencia Cognitiva , Autoimagen , Ondas Encefálicas/fisiología , Estado de Conciencia/fisiología , Humanos , Estimulación Magnética TranscranealRESUMEN
Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Neuronas GABAérgicas/fisiología , Interneuronas/fisiología , Nicotina/farmacología , Refuerzo en Psicología , Área Tegmental Ventral/fisiología , Potenciales de Acción/fisiología , Animales , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas GABAérgicas/efectos de los fármacos , Interneuronas/efectos de los fármacos , Ratones , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/fisiología , Área Tegmental Ventral/efectos de los fármacosRESUMEN
We have described a strategy to analyze the data available on brain genes expression, using the concept of coherent-gene groups controlled by transcription factors (TFs). A hierarchical model of gene-expression patterns during brain development was established that identified the genes assumed to behave as functionally coding. Analysis of the concerned signaling pathways and processes showed distinct temporal gene-expression patterns in relation with neurogenesis/synaptogenesis. We identified the hierarchical tree of TF networks that determined the patterns of genes expressed during brain development. Some 'master TFs' at the top level of the hierarchy regulated the expression of gene groups. Enhanced/decreased activity of a few master TFs may explain paradoxes raised by the genetic determination of autism-spectrum disorders and schizophrenia. Our analysis showed gene-TF networks, common or related, to these disorders that exhibited two maxima of expression, one in the prenatal and the other at early postnatal period of development, consistent with the view that these disorders originate in the prenatal period, develop in the postnatal period, and reach the ultimate neural and behavioral phenotype with different sets of genes regulating each of these periods. We proposed a strategy for drug design based upon the temporal patterns of expression of the concerned TFs. Ligands targeting specific TFs can be designed to specifically affect the pathological evolution of the mutated gene(s) in genetically predisposed patients when administered at relevant stages of brain development.
Asunto(s)
Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Modelos Genéticos , Animales , Encéfalo/crecimiento & desarrollo , Perfilación de la Expresión Génica , Genes , Modelos Estadísticos , Neurogénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The Balb/c strain and the C57BL/6 strain show constitutive differences for tyrosine hydroxylase expression, and noradrenaline (NA) prefrontal transmission. Male mice of these strains also show striking differences in social interaction behaviors, with an increased aggressiveness for the Balb/c strain. To test a potential link between these neurobiological and behavioral parameters, we evaluated the behavioral effects of chronic treatment of mice with BC19, a noreburnamine compound previously known as RU24722, found to modify cell organisation, tyrosine hydoxylase (TH) expression, and its activity into the locus coeruleus (LC). We compared the pharmacological effects between the two strains in social behaviors. Our results show that the emergence of additional TH-expressing (TH+) neurons in the rostral part of the LC of Balb/c mice was associated with an increase in the density of TH+ and noradrenergic (NA+) fibers in the molecular layer in the cingular (Cg1) and prelimbic (PrL) parts of the prefrontal cortex (PFC). BC19 treatment resulted in the near-equalization of the LC number of TH+ neurons and of the density of TH+ and NA+ fibers between both strains. The aggressiveness in Balb/c mice was considerably diminished by BC19 treatment, while the originally non aggressive behavior of C57Bl/6 mice was much less affected by BC19 treatment, despite a moderate increase in some offensive behaviors. In additional control experiments, we checked the effect of BC19 on a separate test for anxiety and assessed the effect of noradrenergic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) mediated lesions in C57BL/6 mice on social behaviors. In the present study we show that the BC19 effect in Balb/c mice was independent of anxiety as measured in the light/dark test and that DSP-4 lesions in C57BL/6 mice produced a robust increase in aggressive social interaction. Altogether, these results show that the noradrenergic system, and particularly its projections to the PFC, strongly modulates aggressive behaviors.
Asunto(s)
Agresión/fisiología , Corteza Prefrontal/enzimología , Conducta Social , Tirosina 3-Monooxigenasa/genética , Agresión/efectos de los fármacos , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Norepinefrina/fisiología , Corteza Prefrontal/efectos de los fármacos , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/metabolismo , Vincamina/análogos & derivados , Vincamina/farmacologíaRESUMEN
RATIONALE: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity beta2-containing nicotinic receptors (beta2*nAChRs) are located. OBJECTIVES: We intend to see which brain circuits are activated when nicotine is given in animals naïve for nicotine and whether the beta2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. MATERIALS AND METHODS: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and beta2 knockout (KO) mice. RESULTS: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, beta2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via alpha7 nicotinic receptors. CONCLUSIONS: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on beta2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice.
Asunto(s)
Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiología , Anestesia , Animales , Química Encefálica/efectos de los fármacos , Diseño de Equipo , Inyecciones Subcutáneas , Imagen por Resonancia Magnética/instrumentación , Masculino , Ratones , Ratones Noqueados , Oxígeno/sangre , Receptores Nicotínicos/efectos de los fármacos , Estimulación QuímicaRESUMEN
The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the beta2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different alpha subunit partners of beta2 (i.e., alpha4 and alpha6), the homo-pentameric alpha7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7-52.6 microg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the beta2, alpha4, alpha6 and alpha7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, beta2-VEC mice with the beta2 subunit re-expressed exclusively in the VTA, alpha4-VEC mice with selective alpha4 re-expression in the VTA, alpha6-VEC mice with selective alpha6 re-expression in the VTA, and alpha7-KO mice promptly self-administer nicotine intravenously, whereas beta2-KO, beta2-VEC in the substantia nigra, alpha4-KO and alpha6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of alpha4beta2- and alpha6beta2-subunit containing nicotinic receptors (alpha4beta2*- and alpha6beta2*-nAChRs), but not alpha7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.
Asunto(s)
Condicionamiento Operante/efectos de los fármacos , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Receptores Nicotínicos/fisiología , Área Tegmental Ventral/metabolismo , Análisis de Varianza , Animales , Autorradiografía/métodos , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Bloqueadores de los Canales de Calcio/farmacocinética , Conotoxinas/farmacocinética , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Isótopos de Yodo/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nicotínicos/deficiencia , Autoadministración/métodos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
It has been suggested that the negative effects associated with nicotine withdrawal promote continued tobacco use and contribute to the high relapse rate of smoking behaviors. Thus, it is important to understand the receptor-mediated mechanisms underlying nicotine withdrawal to aid in the development of more successful smoking cessation therapies. The effects of nicotine withdrawal are mediated through nicotinic acetylcholine receptors (nAChRs); however, the role of nAChRs in nicotine withdrawal remains unclear. Therefore, we used mecamylamine-precipitated, spontaneous, and conditioned place aversion (CPA) withdrawal models to measure physical and affective signs of nicotine withdrawal in various nAChR knockout (KO) mice. beta2, alpha7, and alpha5 nAChR KO mice were chronically exposed to nicotine through surgically implanted osmotic minipumps. Our results show a loss of anxiety-related behavior and a loss of aversion in the CPA model in beta2 KO mice, whereas alpha7 and alpha5 KO mice displayed a loss of nicotine withdrawal-induced hyperalgesia and a reduction in somatic signs, respectively. These results suggest that beta2-containing nAChRs are involved in the affective signs of nicotine withdrawal, whereas non-beta2-containing nAChRs are more closely associated with physical signs of nicotine withdrawal; thus, the nAChR subtype composition may play an important role in the involvement of specific subtypes in nicotine withdrawal.
Asunto(s)
Nicotina , Receptores Nicotínicos/fisiología , Síndrome de Abstinencia a Sustancias , Animales , Condicionamiento Psicológico , Ratones , Ratones Noqueados , Nicotina/administración & dosificación , Subunidades de Proteína/deficiencia , Subunidades de Proteína/fisiología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits alpha5, alpha7, or beta2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, alpha3, which is expressed in all SCG nAChRs, mRNA levels of beta4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, alpha4 declined sharply after birth and was barely detectable in adult animals. alpha5, alpha7, and beta2 subunit message levels also declined, though more slowly and less completely than alpha4. The subunits alpha6 and beta3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas alpha2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation.
Asunto(s)
Neuronas/metabolismo , ARN Mensajero/genética , Receptores Nicotínicos/genética , Ganglio Cervical Superior/crecimiento & desarrollo , Ganglio Cervical Superior/metabolismo , Acetilcolina/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Subunidades de Proteína/genética , ARN Mensajero/metabolismo , Especificidad de la Especie , Ganglio Cervical Superior/citología , Transmisión Sináptica/genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.
Asunto(s)
Anticuerpos/metabolismo , Especificidad de Anticuerpos/inmunología , Inmunohistoquímica/métodos , Neuroquímica/métodos , Subunidades de Proteína/inmunología , Receptores Nicotínicos/inmunología , Acetilcolina/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos/química , Western Blotting , Bungarotoxinas/metabolismo , Corteza Cerebral/anatomía & histología , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Hipocampo/anatomía & histología , Hipocampo/inmunología , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/inmunología , Neuronas/metabolismo , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Receptores Nicotínicos/análisis , Receptores Nicotínicos/genética , Transmisión Sináptica/inmunologíaRESUMEN
BACKGROUND AND PURPOSE: Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (beta2 and alpha5) knockout mice for a comparison with rat brain slice preparations. EXPERIMENTAL APPROACH: Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists. KEY RESULTS: When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations. CONCLUSIONS AND IMPLICATIONS: Targeted deletion of the beta2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.
Asunto(s)
Encéfalo/efectos de los fármacos , Catecolaminas/metabolismo , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/fisiología , Alcaloides/farmacología , Animales , Azocinas/farmacología , Encéfalo/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Yoduro de Dimetilfenilpiperazina/farmacología , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Nicotina/farmacología , Norepinefrina/metabolismo , Subunidades de Proteína/agonistas , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Quinolizinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genética , Tetrodotoxina/farmacologíaRESUMEN
We studied the binding of [(3)H]-epibatidine and [(125)I-]alpha-bungarotoxin, as well as subunit-specific antibodies with purified B lymphocytes of C57Bl/6J mice and found that these cells contained 12,200+/-3200 of alpha4(alpha5)beta2 and 3130+/-750 of alpha7(alpha5beta4) nicotinic acetylcholine receptors per cell. According to flow cytometry data, the highest expression of alpha4(alpha5)beta2 receptors was observed in immature newly generated B lymphocytes of the bone marrow, while the number of alpha7(alpha5beta4) receptors grew up along with the B cell maturation in the spleen. By using alpha4, beta2 or alpha7 knockout and chimera mice, it was shown that both receptor subtypes supported the survival of B cell precursors and increased the size of B-lymphocyte population in the bone marrow. In contrast, propagation of mature B lymphocytes in the spleen was controlled by alpha7-containing subtype only. Moreover, mature B lymphocytes became sensitive to nicotine only in the absence of beta2-containing receptors. Knockout mice had less serum IgG, IgG-producing cells and natural IgG antibodies than their wild-type counterparts, while the absence of beta2-containing receptors resulted in increased B-lymphocyte activation and antibody immune response. The data obtained indicate that nicotinic receptors are involved in regulating B-lymphocyte development and activation, possibly, by affecting expression and/or signaling of CD40, the two subtypes playing different roles.
Asunto(s)
Linfocitos B/fisiología , Activación de Linfocitos , Receptores Nicotínicos/fisiología , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Linfocitos B/metabolismo , Unión Competitiva , Ratones , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Receptores Nicotínicos/inmunología , Receptores Nicotínicos/metabolismoRESUMEN
Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.
Asunto(s)
Encéfalo/fisiología , Vectores Genéticos , Lentivirus/genética , Receptores Nicotínicos/genética , Animales , Conducta Adictiva/genética , Cognición/fisiología , Conducta Exploratoria , Ratones , Ratones Noqueados , Nicotina , Receptores Nicotínicos/deficiencia , Proteínas Recombinantes/metabolismoRESUMEN
RATIONALE: Nicotine addiction is characterized by two distinct behaviors, chronic compulsive self-administration and the induction of a withdrawal syndrome upon cessation of nicotine consumption. OBJECTIVE: To examine if these two processes rely on beta2-containing nicotinic receptors--beta2*nAChRs--we analyzed the behavior of mice lacking these receptors in the two situations. RESULTS: First, we showed that, in contrast to wild-type (WT) mice, beta2-knockout (beta2-/-) mice exhibit no intra-ventral tegmental area (VTA) nicotine self-administration, whereas their ability to self-administer morphine is intact. However, beta2-/- mice showed some sensitivity to locomotor effects of nicotine, implying an effect of the drug on other nicotinic subtypes. Then, we observed that beta2-/- mice exhibited a normal nicotine withdrawal syndrome, i.e., increased levels of rearing and jumping upon precipitated withdrawal. Thus, the beta2*nAChRs are not involved in the behaviors induced by cessation of nicotine consumption. CONCLUSION: Taken together, the present data demonstrated a genetic dissociation of two distinct behavioral patterns associated with nicotine addiction. They further suggested that independent molecular mechanisms underlie these two aspects, offering the possibility of controlling them separately.
Asunto(s)
Nicotina/toxicidad , Receptores Nicotínicos/genética , Refuerzo en Psicología , Síndrome de Abstinencia a Sustancias/genética , Tabaquismo/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfina/administración & dosificación , Nicotina/administración & dosificación , Autoadministración , Área Tegmental Ventral/efectos de los fármacosRESUMEN
Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that beta2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.
Asunto(s)
Cognición/fisiología , Expresión Génica , Nicotina/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Cognición/efectos de los fármacos , Dopamina/metabolismo , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Locomoción/fisiología , Ratones , Morfina/administración & dosificación , Morfina/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nicotina/administración & dosificación , Nicotina/farmacología , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Nicotínicos/deficiencia , Receptores Nicotínicos/genética , Área Tegmental Ventral/citología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/fisiologíaRESUMEN
AIM: To study the ventilatory and arousal/defence responses to hypoxia in newborn mutant mice lacking the beta2 subunit of the nicotinic acetylcholine receptors. METHODS: Breathing variables were measured non-invasively in mutant (n = 31) and wild-type age-matched mice (n = 57) at 2 and 8 days of age using flow barometric whole-body plethysmography. The arousal/defence response to hypoxia was determined using behavioural criteria. RESULTS: On day 2, mutant pups had significantly greater baseline ventilation (16%) than wild-type pups (P < 0.02). Mutant pups had a decreased hypoxic ventilatory declines. Arousal latency was significantly shorter in mutant than in wild-type pups (133 +/- 40 vs. 146 +/- 20 s, respectively, P < 0.026). However, the duration of movement elicited by hypoxia was shorter in mutant than in wild-type pups (14.7 +/- 5.9 vs. 23.0 +/- 10.7 s, respectively, P < 0.0005). Most differences disappeared on P8, suggesting a high degree of functional plasticity. CONCLUSION: The blunted hypoxic ventilatory decline and the shorter arousal latency on day 2 suggested that disruption of the beta2 nicotinic acetylcholine receptors impaired inhibitory processes affecting both the ventilatory and the arousal response to hypoxia during postnatal development.
Asunto(s)
Receptores Nicotínicos/genética , Respiración/genética , Animales , Animales Recién Nacidos , Nivel de Alerta/genética , Nivel de Alerta/fisiología , Temperatura Corporal/fisiología , Peso Corporal/fisiología , Hipoxia/genética , Hipoxia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Pletismografía Total/métodos , Factores de TiempoRESUMEN
Many neurological pathologies are related to misfolded proteins. During folding and assembly in the endoplasmic reticulum, the nicotinic acetylcholine receptor (nAChR) subunits undergo several conformational changes to acquire the ability to bind ligands. After folding and maturation, by mechanisms largely unknown, receptors are exported to the cell surface. We investigated the maturational role of the extracellular C-terminal segment located at the boundary between the extracellular and the transmembrane domains. In the functional chimeric alpha7-5HT3A receptor used as a model system, amino acids from the C-terminal segment were successively deleted or mutated. Upon progressive shortening of the peptide we observed less and less alpha-bungarotoxin binding sites until no sites could be detected when the entire peptide had been deleted (chimera Del 5). Protein synthesis and pentameric assembly were not altered. In Del 5 transfected cells, pentameric receptors present in the endoplasmic reticulum were not detected on the cell surface where Del 5 proteins appeared as patches. With the Del 5 chimera, export of proteins to the cell surface diminished to about half that of wild-type. We propose that the C-terminal segment plays a double role: (i) through an interaction between the penultimate tyrosine residue of the C-terminal segment and the Cys loop of the N-terminal domain, it locks the receptor in a mature alpha-bungarotoxin binding conformation; (ii) this mature conformation, in turn, masks a retention signal present in the first transmembrane segment allowing properly assembled and matured receptors to escape to the cell surface.
Asunto(s)
Membrana Celular/metabolismo , Fragmentos de Péptidos/fisiología , Receptores de Serotonina 5-HT3/biosíntesis , Receptores de Serotonina 5-HT3/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Membrana Celular/genética , Células LLC-PK1 , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Transporte de Proteínas/genética , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Porcinos , Transfección/métodosRESUMEN
Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.
Asunto(s)
Receptores Nicotínicos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Bungarotoxinas/metabolismo , Polaridad Celular , Células Cultivadas , Citoplasma/metabolismo , Células Epiteliales/ultraestructura , Proteínas Fluorescentes Verdes , Hipocampo/citología , Hipocampo/embriología , Células LLC-PK1 , Proteínas Luminiscentes/metabolismo , Neuronas/citología , Neuronas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores de Serotonina/química , Receptores de Serotonina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the alpha4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the alpha4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I alpha-conotoxin MII. These receptors are comprised of the alpha4, beta2 and alpha6 nAChR subunits and non-alpha4, beta2, and alpha6 nAChR subunits. Non-alpha4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and alpha4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that alpha4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.
Asunto(s)
Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Sustancia Negra/efectos de los fármacos , Área Tegmental Ventral/efectos de los fármacos , Animales , Autorradiografía , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Conotoxinas/farmacocinética , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Espacio Extracelular , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microdiálisis , Actividad Motora/efectos de los fármacos , Mutagénesis , Redes Neurales de la Computación , Técnicas de Placa-Clamp , Piridinas/farmacocinética , Receptores Nicotínicos/genética , Sustancia Negra/fisiología , Factores de Tiempo , Área Tegmental Ventral/fisiologíaRESUMEN
We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra-accumbens (Acb) infusion or intraventral tegmental area (intra-VTA) microinjection. The present modes of systemic, intra-Acb and intra-VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra-VTA nicotine administration, whereas unilateral or bilateral intra-Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra-VTA, but not systemic or intra-Acb, nicotine administration caused a long-term (at least 24-h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra-VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra-Acb infusion did not modify any of the mRNAs tested. The long-term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra-VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.
Asunto(s)
Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso , Vías Nerviosas/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Nicotina/farmacología , Núcleo Accumbens/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Área Tegmental Ventral/efectos de los fármacos , Anfetamina/farmacología , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Vías de Administración de Medicamentos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Habituación Psicofisiológica/efectos de los fármacos , Habituación Psicofisiológica/fisiología , Masculino , Proteínas de Transporte de Membrana/genética , Microdiálisis , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Vías Nerviosas/metabolismo , Plasticidad Neuronal/fisiología , Núcleo Accumbens/metabolismo , Terminales Presinápticos/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Trastornos Relacionados con Sustancias/metabolismo , Trastornos Relacionados con Sustancias/fisiopatología , Factores de Tiempo , Tirosina 3-Monooxigenasa/genética , Área Tegmental Ventral/metabolismoRESUMEN
The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. These experiments use mice lacking the beta2 subunit of nicotinic receptors to investigate its role in nicotine discrimination and conditioned taste aversion (CTA). Wild-type and mutant mice were trained either in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement, or in a counterbalanced two-flavour CTA procedure. Rates of lever-pressing of wild-type and mutant mice did not differ. Wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) rapidly and exhibited steep dose-response curves. Mutant mice failed to acquire these nicotine discriminations and exhibited flat dose-response curves. Both wild-type and mutant mice acquired discrimination of nicotine (1.6 mg/kg) although discrimination performance was weak in the mutants. Nicotine initially reduced response rates in wild-type and mutant mice, and tolerance developed to this effect in each genotype. Both genotypes acquired discrimination of morphine (3 mg/kg) with similar degrees of accuracy, and dose-response curves for morphine discrimination in the two genotypes were indistinguishable. Nicotine produced dose-related CTA in both genotypes, but the magnitude of the effect was less in the mutants than in the wild-type controls. It is concluded that nicotinic receptors containing the beta2 subunit play a major role in the discriminative stimulus and taste aversion effects of nicotine that may reflect psychological aspects of tobacco dependence. Such receptors appear to have a less crucial role in the response-rate, reducing effects of nicotine and in nicotine tolerance.