RESUMO
Overactive bladder is a troublesome disease that affects 15% of the population in developed countries. Since pharmacotherapy of this condition is frequently associated with side effects, the better tolerated drugs are being searched for. The main objective of our study was to check whether activation of the atypical cannabinoid receptor GPR55 would normalize the changes in cystometric, cardiovascular and biochemical parameters in the hypertensive female Wistar-Kyoto rats presenting the symptoms of overactive bladder accompanied by inflammation and oxidative damage in the urinary tracts. A 14-day intra-arterial administration of O-1602 (0.25 mg/kg/day), a potent agonist of GRP55 receptors, was able to abolish the signs of detrusor overactivity, inflammation and oxidative damage in the urinary bladder of the spontaneously hypertensive animals. Moreover, it increased their heart rate, reduced the mean blood pressure, and normalized the levels of several proteins that play a significant role in the proper functioning of the urinary bladder (i.e., calcitonin gene related peptide, organic cation transporter 3, extracellular signal-regulated kinase 1/2, vesicular acetylcholine transporter, RhoA). Based on the outcomes of our experiments, the atypical cannabinoid receptor GPR55 has emerged as a potential drug target for the treatment of overactive bladder in female subjects. It could be particularly attractive in the cases in which this condition is accompanied with elevated blood pressure, though further studies on this subject are needed.
Assuntos
Canabidiol/análogos & derivados , Hipertensão/tratamento farmacológico , Receptores Acoplados a Proteínas G/agonistas , Bexiga Urinária Hiperativa/tratamento farmacológico , Bexiga Urinária/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Canabidiol/farmacologia , Canabidiol/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Hipertensão/fisiopatologia , Fator 3 de Transcrição de Octâmero/fisiologia , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Canabinoides/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/fisiopatologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.
Assuntos
Pulmão/embriologia , Pulmão/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Animais , Embrião de Galinha , Galinhas , Gânglios/embriologia , Mamíferos/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Sistema Nervoso Parassimpático/embriologia , Alvéolos Pulmonares/embriologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Depression is extremely harmful to modern society. Despite its complex spectrum of symptoms, previous studies have mostly focused on the monaminergic system in search of pharmacological targets. However, other neurotransmitter systems have also been linked to the pathophysiology of depression. In this study, we provide evidence for a role of the cholinergic system in depressive-like behavior of female mice. We evaluated mice knockdown for the vesicular acetylcholine transporter (VAChT KD mice), which have been previously shown to exhibit reduced cholinergic transmission. Animals were subjected to the tail suspension and marble burying tests, classical paradigms to assess depressive-like behaviors and to screen for novel antidepressant drugs. In addition, brain levels of serotonin and dopamine were measured by high performance liquid chromatography. We found that female homozygous VAChT KD mice spent less time immobile during tail suspension and buried less marbles, indicating a less depressive phenotype. These differences in behavior were reverted by central, but not peripheral, acetylcholinesterase inhibition. Moreover, female homozygous VAChT KD mice exhibited higher levels of dopamine and serotonin in the striatum, and increased dopamine in the hippocampus. Our study thus shows a connection between depressive-like behaviors and the cholinergic system, and that the latter interacts with the monoaminergic system.
Assuntos
Depressão/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Acetilcolina/metabolismo , Animais , Antidepressivos/farmacologia , Encéfalo/metabolismo , Corpo Estriado/metabolismo , Transtorno Depressivo/fisiopatologia , Modelos Animais de Doenças , Dopamina/metabolismo , Dopamina/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Neurotransmissores/farmacologia , Serotonina/metabolismo , Serotonina/farmacologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox ). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.
Assuntos
Transtornos Neurológicos da Marcha/genética , Neurônios/fisiologia , Núcleo Tegmental Pedunculopontino/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Animais , Transtornos Neurológicos da Marcha/psicologia , Deleção de Genes , Força da Mão , Deficiências da Aprendizagem/genética , Locomoção , Masculino , Camundongos , Transtornos das Habilidades Motoras/genética , Mutação/genética , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/fisiologia , Núcleo Tegmental Pedunculopontino/citologia , Equilíbrio Postural , Desempenho Psicomotor , Tegmento Mesencefálico/citologia , Tegmento Mesencefálico/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
BACKGROUND: Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). METHODS: Chat-ChR2-EYFP (VAChTHyp) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1G93A), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. RESULTS: We show that VAChT is elevated in the spinal cord and at NMJs of VAChTHyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChTHyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChTHyp mice compared to control mice. While the development of NMJs was not affected in VAChTHyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChTHyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChTHyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChTHyp mice. In the SOD1G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice. CONCLUSIONS: The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.
Assuntos
Acetilcolina/metabolismo , Envelhecimento , Esclerose Lateral Amiotrófica/fisiopatologia , Junção Neuromuscular/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Potenciais Pós-Sinápticos em Miniatura , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Junção Neuromuscular/metabolismo , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Análise de Sobrevida , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
BACKGROUND: Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina. METHODS & RESULTS: A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5) deletion of VAChT (VAChTSix3-Cre-flox/flox) and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses. SIGNIFICANCE: This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.
Assuntos
Acetilcolina/fisiologia , Neurotransmissores/fisiologia , Retina/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Animais , Western Blotting , Eletrorretinografia , Deleção de Genes , Masculino , Camundongos , Camundongos Knockout , Nervo Óptico/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/fisiologia , Tomografia de Coerência Óptica , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.
Assuntos
Edema/fisiopatologia , Pulmão/patologia , Pneumonia/fisiopatologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Acetilcolina/genética , Acetilcolina/metabolismo , Animais , Western Blotting , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Edema/etiologia , Técnicas Imunoenzimáticas , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Pneumonia/etiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aging is marked by progressive impairments in the process of adult neurogenesis and spatial memory performance. The underlying mechanisms for these impairments have not been fully established; however, they may coincide with decline of cholinergic signaling in the hippocampus. This study investigates whether augmenting cholinergic neurotransmission, by enhancing the expression of the vesicular acetylcholine transporter (VAChT), influences the age-related decline in the development of newborn hippocampal cells and spatial memory. We found that enhanced VAChT expression in the hippocampus of mice contributes to lifelong increases in the dendritic complexity of newborn neurons. Furthermore, enhanced VAChT expression improved memory acquisition through an increased use of spatially precise search strategies in the Morris water maze through the course of the aging process. These data suggest that VAChT overexpression contributes to increases in dendritic complexity and improved spatial memory during aging.
Assuntos
Dendritos/fisiologia , Hipocampo/citologia , Memória Espacial , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Envelhecimento , Animais , Células Cultivadas , Neurônios Colinérgicos/fisiologia , Expressão Gênica , Hipocampo/fisiologia , Hipocampo/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Neurogênese , Memória Espacial/fisiologia , Transmissão Sináptica , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.
Assuntos
Atenção/fisiologia , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Cognição/fisiologia , Resistência Física/genética , Resistência Física/fisiologia , Animais , Ansiedade/psicologia , Western Blotting , Channelrhodopsins , Imunofluorescência , Teste de Tolerância a Glucose , Força da Mão/fisiologia , Elevação dos Membros Posteriores , Aprendizagem em Labirinto/fisiologia , Metabolismo/genética , Metabolismo/fisiologia , Camundongos , Camundongos Transgênicos , Sistema Nervoso Parassimpático/fisiologia , Reação em Cadeia da Polimerase , Equilíbrio Postural/fisiologia , Desempenho Psicomotor/fisiologia , Tempo de Reação/fisiologia , Natação/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.
Assuntos
Proteínas de Homeodomínio/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Neurotransmissores/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Contagem de Células , Feminino , Feto , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação/fisiologia , Gravidez , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/genética , Somatostatina/genética , Somatostatina/fisiologia , Gânglio Estrelado/citologia , Gânglio Estrelado/crescimento & desenvolvimento , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/embriologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/fisiologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
The exocytotic release of neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H(+) Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1).
Assuntos
Regulação da Expressão Gênica/fisiologia , Modelos Moleculares , Família Multigênica/genética , Doenças do Sistema Nervoso/metabolismo , Conformação Proteica , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Animais , Monoaminas Biogênicas/metabolismo , Transporte Biológico/fisiologia , Caenorhabditis elegans , Drosophila , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Modelos Biológicos , Mutação/genética , Fosforilação , Filogenia , Proteínas Vesiculares de Transporte de Acetilcolina/biossíntese , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.
Assuntos
Fusão de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clatrina/fisiologia , Clatrina/ultraestrutura , Endocitose/fisiologia , Exocitose/fisiologia , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Microscopia de Interferência/métodos , Células PC12/fisiologia , Ratos , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Acetilcolina/ultraestruturaRESUMO
Cholinergic input modulates excitability of motoneurons and plays an important role in the control of locomotion in both intact and spinalized animals. However, spinal cord transection in adult rats affects cholinergic innervation of only some hindlimb motoneurons, suggesting that specificity of this response is related to functional differences between motoneurons. Our aim was therefore to compare cholinergic input to motoneurons innervating the soleus (Sol) and tibialis anterior (TA) motoneurons following spinal cord transection at a low-thoracic level. The second aim was to investigate whether deficits in cholinergic input to these motoneurons could be modified by locomotor training. The Sol and TA motoneurons were identified by retrograde labelling with fluorescent dyes injected intramuscularly. Cholinergic terminals were detected using anti-vesicular acetylcholine transporter (VAChT) antibody. Overall innervation of motoneurons was evaluated with anti-synaptophysin antibody. After spinalization we found a decrease in the number of VAChT-positive boutons apposing perikarya of the Sol (to 49%) but not TA motoneurons. Locomotor training, resulting in moderate functional improvement, partly reduced the deficit in cholinergic innervation of Sol motoneurons by increasing the number of VAChT-positive boutons. However, the optical density of VAChT-positive boutons terminating on various motoneurons, which decreased after spinalization, continued to decrease despite the training, suggesting an impairment of acetylcholine availability in the terminals. Different effects of spinal cord transection on cholinergic innervation of motoneurons controlling the ankle extensor and flexor muscles point to different functional states of these muscles in paraplegia as a possible source of activity-dependent signaling regulating cholinergic input to the motoneurons.
Assuntos
Neurônios Colinérgicos/fisiologia , Locomoção/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/fisiologia , Acetilcolina/fisiologia , Animais , Masculino , Músculo Esquelético/inervação , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Medula Espinal/cirurgia , Tarso Animal/inervação , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.
Assuntos
Acetilcolina/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/fisiologia , Acetilcolina/fisiologia , Alcenos/farmacologia , Animais , Relação Dose-Resposta a Droga , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/fisiologia , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Fisostigmina/farmacologia , Piperidinas/farmacologia , Receptores Colinérgicos/fisiologia , Vesículas Secretórias/fisiologia , Transdução de Sinais/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-ß-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.
Assuntos
Acetilcolina/metabolismo , Endocitose/fisiologia , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , beta-Ciclodextrinas/farmacologia , Animais , Endocitose/efeitos dos fármacos , Masculino , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/antagonistas & inibidoresRESUMO
The parasympathetic vasodilatory fibres are known to innervate vessels in a rat masseter muscle via both cholinergic and non-cholinergic mechanisms. However, the non-cholinergic mechanisms are still unclear. Recently, vasoactive intestinal polypeptide (VIP) was convincingly shown to be involved in the parasympathetic vasodilatation in orofacial areas, such as submandibular glands and lower lip. However, very little is known about the rat masseter muscle. The present study was designed in the rat masseter muscle to assess (1) whether the parasympathetic nerve innervating vessels have VIP immunoreactivities, (2) whether intravenous administration of VIP induces the vasodilatation, and (3) effects of selective VIP receptor antagonist ([4Cl-d-Phe(6), Leu(17)] VIP) in the presence or absence of atropine on the parasympathetic vasodilatation. The VIP immunoreactivities were found at two sites of the parasympathetic otic ganglion and nerve fibres located around vessels. The intravenous administration of VIP induced the vasodilatation, and [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the vasodilatation evoked by VIP administration. The parasympathetic vasodilatation was not inhibited by [4Cl-d-Phe(6), Leu(17)] VIP. However, treatment with [4Cl-d-Phe(6), Leu(17)] VIP markedly decreased the parasympathetic vasodilatation when [4Cl-d-Phe(6), Leu(17)] VIP was administered together with atropine. These results suggest that (1) VIP exists in the postganglionic parasympathetic nerve innervating the vessels in the masseter muscle, (2) the intravenous administration of VIP induces the vasodilatation in the masseter muscle, and (3) VIP may be involved in the parasympathetic vasodilatation in the masseter muscle when muscarinic cholinergic receptors are deactivated by either atropine or the suppression of the ACh release.
Assuntos
Músculo Masseter/irrigação sanguínea , Músculo Masseter/inervação , Fibras Parassimpáticas Pós-Ganglionares/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Vasodilatação/fisiologia , Animais , Atropina/farmacologia , Imunofluorescência , Gânglios Parassimpáticos/fisiologia , Fluxometria por Laser-Doppler , Masculino , Fibras Parassimpáticas Pós-Ganglionares/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Ratos , Ratos Wistar , Receptores de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Traumatic brain injury (TBI) is one of the leading causes of death and disability. Cognitive deficits are believed to be connected with impairments of the cholinergic system. The present study was conducted to evaluate the cholinergic system in a model of focal brain injury with special attention to the time course of posttraumatic events in critical brain regions. Three groups of male Sprague-Dawley rats (post-TBI survival time: 2 h, 24 h and 72 h) were subjected to sham-operation (control) or controlled cortical impact injury. Receptor densities were determined on frozen ipsilateral sagittal brain sections with [(3)H]epibatidine (nicotinic acetylcholine receptors) and [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was evaluated with (-)[(3)H]vesamicol. Compared to control, vAChT was lowered (up to 50%) at each time point after trauma, with reductions in olfactory tubercle, basal forebrain, motor cortex, putamen, thalamic and hypothalamic areas and the gigantocellular reticular nucleus. Time-dependent reductions of about 20% of nAChR-density in the thalamus, hypothalamus, olfactory tubercle, gigantocellular reticular nucleus and motor cortex were observed post-TBI at 24 and 72 h. The same brain regions showed reductions of mAChR at 24 and 72 h after trauma with additional decreases in the corpus callosum, basal forebrain and anterior olfactory nucleus. In conclusion, cholinergic markers showed significant time-dependent impairments after TBI. Considering the role of the cholinergic system for cognitive processes in the brain, it seems likely that these impairments contribute to clinically relevant cognitive deficits.
Assuntos
Lesões Encefálicas/fisiopatologia , Receptores Colinérgicos/fisiologia , Animais , Autorradiografia , Encéfalo/patologia , Lesões Encefálicas/patologia , Colina/metabolismo , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/fisiologia , Receptores Nicotínicos/fisiologia , Fatores de Tempo , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaAssuntos
Neurotransmissores/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Neurotransmissores/fisiologia , Animais , Humanos , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Proteínas Vesiculares de Transporte de Glutamato/fisiologia , Proteínas Vesiculares de Transporte de Monoamina/fisiologiaAssuntos
Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Fibras Colinérgicas/fisiologia , Corpo Estriado/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Acetilcolina/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Ácido Glutâmico/fisiologia , Camundongos , Camundongos Knockout , Modelos Neurológicos , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
In this study, we address why metabotropic and ionotropic cholinergic signaling pathways are used to facilitate motor behaviors. We demonstrate that a G alpha(q)-coupled muscarinic acetylcholine receptor (mAChR) signaling pathway enhances nicotinic acetylcholine receptor (nAChR) signaling to facilitate the insertion of the Caenorhabditis elegans male copulatory spicules into the hermaphrodite during mating. Previous studies showed that ACh (acetylcholine) activates nAChRs on the spicule protractor muscles to induce the attached spicules to extend from the tail. Using the mAChR agonist Oxo M (oxotremorine M), we identified a GAR-3(mAChR)-G alpha(q) pathway that promotes protractor muscle contraction by upregulating nAChR signaling before mating. GAR-3(mAChR) is expressed in the protractor muscles and in the spicule-associated SPC and PCB cholinergic neurons. However, ablation of these neurons or impairing cholinergic transmission reduces drug-induced spicule protraction, suggesting that drug-stimulated neurons directly activate muscle contraction. Behavioral analysis of gar-3 mutants indicates that, in wild-type males, GAR-3(mAChR) expression in the SPC and PCB neurons is required for the male to sustain rhythmic spicule muscle contractions during attempts to breach the vulva. We propose that the GAR-3(mAChR)/G alpha(q) pathway sensitizes the spicule neurons and muscles before and during mating so that the male can respond to hermaphrodite vulva efficiently.