RESUMO
The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 107 CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.
Assuntos
Cápsulas Bacterianas/metabolismo , Viabilidade Microbiana , Microglia/microbiologia , Fagocitose , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/metabolismo , Animais , Cápsulas Bacterianas/imunologia , Encéfalo/microbiologia , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Imuno-Histoquímica , Dose Letal Mediana , Meningites Bacterianas , Camundongos , Microglia/imunologia , Microscopia Eletrônica de Transmissão , Streptococcus pneumoniae/imunologia , Análise de Sobrevida , Virulência , Fatores de Virulência/imunologiaRESUMO
alpha6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of alpha6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of alpha6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of alpha6* nAChR in the mesostriatal system is heterogeneous, with (non-alpha4)alpha6beta2* being predominant in the mesolimbic pathway and alpha4alpha6beta2* in the nigrostriatal pathway. We verified whether alpha6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists alpha-conotoxin MII (CntxMII) (alpha3/alpha6beta2* selective) or alpha-conotoxin PIA (CntxPIA) (alpha6beta2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the alpha6beta2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that alpha6beta2*-selective compounds capable of crossing the blood-brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.
Assuntos
Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologia , Animais , Conotoxinas/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiologia , Dopamina/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Antagonistas Nicotínicos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Ratos , Ratos Sprague-Dawley , Reforço Psicológico , AutoadministraçãoRESUMO
We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [(3)H]noradrenaline ([(3)H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K(+). NMDA -evoked [(3)H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [(3)H]NA overflow elicited by 100 muM AMPA, 1 muM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn(2+) ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.
Assuntos
Meio Ambiente , Hipocampo/metabolismo , Norepinefrina/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Receptores Pré-Sinápticos/fisiologia , Regulação para Cima/fisiologia , Animais , Feminino , Hipocampo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologiaRESUMO
Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo-interpeduncular (Hb-IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (+/+) and beta2 knock-out (-/-) mice to establish that the Hb and IPn contain significant beta2* and beta4* populations of nAChR receptors (each of which is heterogeneous). The beta4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (alpha2beta2*, alpha4beta3beta2*, alpha3beta3beta4*, alpha6beta3beta4*). Our studies on IPn synaptosomes obtained from +/+ and alpha2, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4(-/-) mice show that only the alpha3beta4 and alpha3beta3beta4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in beta3(-/-) mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and alpha3beta4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the beta3 subunit may be important for transporting the alpha3beta4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb-IPn system and identify alpha3beta4 and alpha3beta3beta4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.
Assuntos
Acetilcolina/metabolismo , Habenula/metabolismo , Mesencéfalo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Habenula/citologia , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Vias Neurais/citologia , Vias Neurais/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Transmissão Sináptica/genética , Tegmento MesencefálicoRESUMO
Synapsin I (SynI) and synapsin II (SynII) are major synaptic vesicle (SV) proteins that function in the regulation of the availability of SVs for release in mature neurons. SynI and SynII show a high level of sequence similarity and share many functions in vivo, although distinct physiological roles for the two proteins have been proposed. Both SynI(-/-) and SynII(-/-) mice have a normal lifespan, but exhibit a decreased number of SVs and synaptic depression upon high-frequency stimulation. Because of the role of the synapsin proteins in synaptic organization and plasticity, we studied the long-lasting effects of synapsin deletion on the phenotype of SynI(-/-) and SynII(-/-) mice during aging. Both SynI(-/-) and SynII(-/-) mice displayed behavioural defects that emerged during aging and involved emotional memory in both mutants, and spatial memory in SynII(-/-) mice. These abnormalities, which were more pronounced in SynII(-/-) mice, were associated with neuronal loss and gliosis in the cerebral cortex and hippocampus. The data indicate that SynI and SynII have specific and non-redundant functions, and that synaptic dysfunctions associated with synapsin mutations negatively modulate cognitive performances and neuronal survival during senescence.
Assuntos
Senescência Celular/fisiologia , Transtornos Cognitivos/metabolismo , Transtornos da Memória/fisiopatologia , Sinapsinas/metabolismo , Animais , Comportamento Animal/fisiologia , Hipocampo/metabolismo , Hipocampo/patologia , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Neocórtex/metabolismo , Neocórtex/patologia , Neurônios/patologia , Neurônios/fisiologia , Testes Neuropsicológicos , Sinapsinas/genéticaRESUMO
Pharmacological activation of nicotinic acetylcholine receptors (nAChRs) exerts neuroprotective effects in cultured neurons and the intact animal. Much less is known about a physiological protective role of nAChRs. To understand whether endogenous activation of beta2* nAChRs contributes to the maintenance of the functional and morphological integrity of neural tissue, adult beta2-/- mice were subjected to in vivo challenges that cause neurodegeneration and cognitive impairment (intrahippocampal injection of the excitotoxin quinolinic acid), or neuroprotection and cognitive potentiation (2-month exposure to an enriched environment). The excitotoxic insult caused an increased deficit in the Morris water maze learning curve and increased loss of hippocampal pyramidal cells in beta2-/- mice. Exposure to an enriched environment improved performance in contextual and cued fear conditioning and object recognition tests in beta2+/+, whereas the improvement was absent in beta2-/- mice. In addition, beta2+/+, but not beta2-/-, mice exposed to an enriched environment showed a significant hypertrophy of the CA1/3 regions. Thus, lack of beta2* nAChRs increased susceptibility to an excitotoxic insult and diminished the positive effects of an enriched environment. These results may be relevant to understanding the pathophysiological consequences of the marked decrease in nAChRs that occurs in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
Assuntos
Comportamento Animal/efeitos dos fármacos , Planejamento Ambiental , Hipocampo/efeitos dos fármacos , Neurotoxinas/administração & dosagem , Ácido Quinolínico/administração & dosagem , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , Animais , Comportamento Animal/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotoxinas/toxicidade , Ligação Proteica/genética , Ácido Quinolínico/toxicidade , Receptores Nicotínicos/fisiologiaRESUMO
Neuronal cholinergic nicotinic receptors (nAChRs) are a heterogeneous class of cationic channels that are widely distributed in the nervous system that have specific functional and pharmacological properties. They consist of homologous subunits encoded by a large multigene family, and their opening is physiologically controlled by the acetylcholine neurotransmitter or exogenous ligands such as nicotine. Their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding receptor function in the nervous system and discovering new subtype-selective drugs. We will review rodent brain subtypes by discussing their subunit composition, pharmacology and localisation and, when possible, comparing them with the same subtypes present in the brain of other mammalian species or chick. In particular, we will focus on the nAChRs present in the visual pathway (retina, superior colliculus and nucleus geniculatus lateralis), in which neurons express most, if not all, nAChR subunits. In addition to the major alpha4beta2 and alpha7 nAChR subtypes, the visual pathway selectively expresses subtypes with a complex subunit composition. By means of ligand binding and immunoprecipitation and immunopurification experiments on tissues obtained from control and lesioned rats, and wild-type and nAChR subunit knockout mice, we have qualitatively and quantitatively identified, and pharmacologically characterised, the multiple complex native subtypes containing up to four different subunits.
Assuntos
Química Encefálica , Receptores Nicotínicos/classificação , Animais , Humanos , Subunidades Proteicas , Receptores Nicotínicos/análise , Receptores Nicotínicos/química , Vias Visuais/químicaRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of cation channels widely distributed in the brain, whose subunit composition and biophysical properties vary depending on the subtype and the area of the brain in which they are found. Brain nAChRs are also the target of nicotine, the most widespread drug of abuse. Chronic nicotine exposure differentially affects the number, subunit composition, stoichiometry and functional state of some nAChR subtypes, leaving others substantially unaffected. In this review, we will summarise recent data concerning the nAChR subtypes expressed in the CNS, and how they are regulated by means of chronic nicotine and/or nicotinic drugs. We will particularly focus on the possible mechanisms involved in the up-regulation of nAChRs.
Assuntos
Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Encéfalo/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptores Nicotínicos/química , Receptores Nicotínicos/genéticaRESUMO
RATIONALE: Several studies have suggested that nicotine treatment can modulate the behavioral and neurochemical responses to other psychostimulants, such as cocaine. OBJECTIVES: The current study examined the hypothesis that nicotinic acetylcholine receptor (nAChR) blockade influences the ability of cocaine to elicit increases in extracellular dopamine levels. MATERIALS AND METHODS: Pharmacological studies using nicotinic antagonists as well as genetic inactivation of beta2* nAChRs were used to determine the effect of nAChR blockade on dopamine levels in ventral striatum elicited by acute or repeated administrations of cocaine in mice. RESULTS: Administration of mecamylamine (a general nicotinic antagonist that is not highly selective for individual nAChR subtypes) or co-administration of methyllycaconitine (a more selective antagonist of alpha7* nAChRs) with dihydro-beta-erythroidine (a more selective antagonist of beta2* nAChRs and other heteromeric nAChR subtypes) prevented sensitization of cocaine-elicited increases in extracellular DA levels in the ventral striatum in wild-type mice. In contrast, neither of the more specific antagonists alone was effective in preventing sensitization. Finally, methyllycaconitine administration prevents sensitization in beta2-/- mice but not in beta2+/+ or wild-type mice. CONCLUSIONS: These data indicate that inhibition of both alpha7* and beta2* nAChRs is necessary to prevent development of sensitization of cocaine-elicited increases in extracellular dopamine levels in the ventral striatum of mice.
Assuntos
Aconitina/análogos & derivados , Gânglios da Base/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Dopamina/metabolismo , Líquido Extracelular/efeitos dos fármacos , Mecamilamina/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Aconitina/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been implicated in the activity-dependent development and plasticity of retina and the refinement of retinal projections. Pharmacological and functional studies have also indicated that different presynaptic nAChRs can have a modulatory function in retinotectal synapses. We biochemically and pharmacologically identified the multiple nAChR subtypes expressed on retinal afferents of the superior colliculus (SC) and lateral geniculate nucleus (LGN). We found that the alpha6beta2(*) and alpha4(nonalpha6)beta2(*) nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of alpha2alpha6beta2(*) and alpha3beta2(*) subtypes, whereas the SC contains a relatively large population of a new native subtype, the alpha3beta2(alpha5/beta3) nAChR. This subtype binds the alpha-conotoxin MII with an affinity 50 times lower than that of the native alpha6beta2(*) subtype. Studies of tissues obtained from eye-enucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC alpha6beta2(*), alpha4alpha6beta2(*), and alpha3beta2(*) (approximately 45, 35, and 20%, respectively), in LGN, alpha4alpha6beta2(*), alpha6beta2(*), alpha4beta2(*), alpha2alpha6beta2(*), and alpha3beta2(*) (approximately 40, 30, 20, 5, and 5%, respectively). In both regions, more than 50% of nAChRs were not expressed by retinal afferents and belonged to the alpha4beta2(*) (90%) or alpha4alpha5beta2(*) (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and alpha4, alpha6, and beta3 knockout mice confirmed and extended the data obtained in rat tissue and allowed a comprehensive dissection of the composition of nAChR subtypes present in this retinorecipient area.
Assuntos
Corpos Geniculados/metabolismo , Neurônios Aferentes/metabolismo , Receptores Nicotínicos/metabolismo , Colículos Superiores/metabolismo , Sequência de Aminoácidos , Animais , Imunoprecipitação , Ligantes , Masculino , Dados de Sequência Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/classificação , Retina/metabolismoRESUMO
Adult hippocampal neurogenesis declines with age in parallel with decreased performance on a variety of hippocampal-dependent tasks. We measured the rate of cellular proliferation in the hippocampus of mice lacking the beta 2-subunit of the nicotinic acetylcholine receptor (beta 2-/- mice) at three ages: young adult (3 months old), fully adult (7-10 months old), and aged (22-24 months old). Consistent with previous studies, we observed an age-related decline in hippocampal proliferation in both groups. However, in fully adult beta 2-/- mice a 43% reduction of granule cell proliferation was detected compared to age-matched controls. This was accompanied by a significant decrease in dentate gyrus area/section and the length of the granule cell layer in beta 2-/- mice. These alterations were not the result of a change in plasma corticosterone levels or expression of the neurotrophic factor BDNF in the dentate gyrus, two known regulators of hippocampal cell proliferation. Similarly, there was no increase in gliosis, abnormal myelination, or apoptotic cell death in the beta 2-/- animals, although there was a significant shift in the location of apoptotic cells in the dentate gyrus indicative of a change in neuronal survival. These results suggest that the beta 2-subunit containing nicotinic acetylcholine receptors play an important role in regulating cell proliferation in the hippocampus and that endogenous acetylcholine may act to oppose the negative effects of normal aging and stress on cellular proliferation.
Assuntos
Hipocampo/citologia , Hipocampo/fisiologia , Neurônios/citologia , Receptores Nicotínicos/genética , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Apoptose , Divisão Celular/fisiologia , Feminino , Gliose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologiaRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) expressed on mesostriatal dopaminergic neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Using immunoprecipitation and ligand-binding techniques, we have shown that both alpha6beta2* and alpha4(nonalpha6)beta2* nAChRs are expressed in the caudate-putamen and that only alpha6* nAChRs can bind alpha-conotoxin MII and methyllycaconitine with affinities of 1.3 and 40 nm, respectively. Further studies performed on 6-hydroxydopamine-lesioned striatum led to the identification of nAChR subtypes selectively expressed on dopaminergic terminals [alpha4alpha5beta2, alpha4alpha6beta2(beta3), and alpha6beta2(beta3)], nondopaminergic neuronal structures (alpha2alpha4beta2), or both structures (alpha4beta2). The identification of the nAChRs expressed on striatal dopaminergic terminals opens up the possibility of developing selective nAChR ligands active on dopaminergic systems and associated diseases, such as Parkinson's disease.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Subunidades Proteicas , Receptores Nicotínicos/biossíntese , Animais , Ligação Competitiva/efeitos dos fármacos , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Ligantes , Masculino , Agonistas Nicotínicos/farmacocinética , Antagonistas Nicotínicos/farmacocinética , Oxidopamina , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/análise , Receptores Nicotínicos/classificação , Organismos Livres de Patógenos EspecíficosRESUMO
Impairment in cholinergic systems is a highly consistent finding in human dementia. Among cholinergic markers, marked decreases in nicotine binding have been most consistently observed in the telencephalic regions of demented patients and are thought to contribute to the cognitive deficits associated with ageing and age-related neurodegenerative diseases. New evidence that the cholinergic system has a specific pathogenic role in the neurodegenerative alterations of aged and, especially, demented patients is fast accumulating. Both in vivo and in culture, nicotine protects striatal, hippocampal and cortical neurons against the neurotoxicity induced by excitotoxic amino acids as well as the toxicity caused by beta-amyloid, the major component of senile plaques. Further support for the implication of nicotinic receptors in brain ageing is come from recent studies on transgenic animals lacking nicotinic receptor subtypes, which shed light on the mechanisms of nicotine neuroprotection and neurotoxicity.