RESUMEN
OBJECTIVE: Arterial stenosis is a significant cardiovascular disease requiring accurate estimation of the pressure gradients for determining hemodynamic significance. In this paper, we propose Generalized Bernoulli Equation (GBE) utilizing interpolated-based method to estimate relative pressures using streamlines and pathlines from 4D Flow MRI. METHODS: 4D Flow MRI data in a stenotic phantom model and computational fluid dynamics simulated velocities generated under identical flow conditions were processed by Generalized Bernoulli Equation (GBE), Reduced Bernoulli Equations (RBE), as well as the Simple Bernoulli Equation (SBE) which is clinically prevalent. Pressures derived from 4D flow MRI and noise corrupted CFD velocities were compared with pressures generated directly with CFD as well as pressures obtained using Millar catheters under identical flow conditions. RESULTS: It was found that SBE and RBE methods underestimated the relative pressure for lower flow rates while overestimating the relative pressure at higher flow rates. Specifically, compared to the reference pressure, SBE underestimated the maximum relative pressure by 22[Formula: see text] for a pulsatile flow data with peak flow rate [Formula: see text] and overestimated by around 40[Formula: see text] when [Formula: see text]. In contrast, for GBE method the relative pressure values were overestimated by 15[Formula: see text] with [Formula: see text]and around 10[Formula: see text] with [Formula: see text]. CONCLUSION: GBE methods showed robust performance to additive image noise compared to other methods. Our findings indicate that GBE pressure estimation over pathlines attains the highest level of accuracy compared to GBE over streamlines, and the SBE and RBE methods.
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Imagen por Resonancia Magnética , Enfermedades Vasculares , Constricción Patológica/diagnóstico por imagen , Hemodinámica , Humanos , Hidrodinámica , Flujo PulsátilRESUMEN
Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.
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Acetilcolina/metabolismo , Unión Neuromuscular/metabolismo , Acetilación , Acetilcolina/agonistas , Animales , Carbacol/farmacología , Diferenciación Celular , Agonistas Colinérgicos/farmacología , Ratones , Unión Neuromuscular/citología , Unión Neuromuscular/efectos de los fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMEN
Cerebellar Purkinje neurons maintain high firing rates but their synaptic terminals depress only moderately, raising the question of how vesicle depletion is minimized. To identify mechanisms that limit synaptic depression, we evoked 100 Hz trains of GABAergic inhibitory postsynaptic currents (IPSCs) in cerebellar nuclear neurons by stimulating Purkinje axons in mouse brain slices. The paired-pulse ratio (IPSC(2)/IPSC(1)) of the total IPSC was approximately 1 and the steady-state ratio (IPSC(20)/IPSC(1)) was approximately 0.5, suggesting a high response probability of postsynaptic receptors, without an unusually high release probability. Three-dimensional electron microscopic reconstructions of Purkinje boutons revealed multiple active zones without intervening transporters, suggestive of "spillover"-mediated transmission. Simulations of boutons with 10-16 release sites, in which transmitter from any site can reach all receptors opposite the bouton, replicated multiple-pulse depression during normal, high, and low presynaptic Ca influx. These results suggest that release from multiple-site boutons limits depletion-based depression, permitting prolonged, high-frequency inhibition at corticonuclear synapses.
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Núcleos Cerebelosos/fisiología , Terminales Presinápticos/fisiología , Células de Purkinje/fisiología , Transmisión Sináptica/fisiología , Animales , Axones/fisiología , Simulación por Computador , Estimulación Eléctrica , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Modelos Neurológicos , Inhibición Neural/fisiología , Células de Purkinje/ultraestructura , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Biochemical and genetic data suggest that synaptotagmin-2 functions as a Ca2+ sensor for fast neurotransmitter release in caudal brain regions, but animals and/or synapses lacking synaptotagmin-2 have not been examined. We have now generated mice in which the 5' end of the synaptotagmin-2 gene was replaced by lacZ. Using beta-galactosidase as a marker, we show that, consistent with previous studies, synaptotagmin-2 is widely expressed in spinal cord, brainstem, and cerebellum, but is additionally present in selected forebrain neurons, including most striatal neurons and some hypothalamic, cortical, and hippocampal neurons. Synaptotagmin-2-deficient mice were indistinguishable from wild-type littermates at birth, but subsequently developed severe motor dysfunction, and perished at approximately 3 weeks of age. Electrophysiological studies in cultured striatal neurons revealed that the synaptotagmin-2 deletion slowed the kinetics of evoked neurotransmitter release without altering the total amount of release. In contrast, synaptotagmin-2-deficient neuromuscular junctions (NMJs) suffered from a large reduction in evoked release and changes in short-term synaptic plasticity. Furthermore, in mutant NMJs, the frequency of spontaneous miniature release events was increased both at rest and during stimulus trains. Viewed together, our results demonstrate that the synaptotagmin-2 deficiency causes a lethal impairment in synaptic transmission in selected synapses. This impairment, however, is less severe than that produced in forebrain neurons by deletion of synaptotagmin-1, presumably because at least in NMJs, synaptotagmin-1 is coexpressed with synaptotagmin-2, and both together mediate fast Ca2+-triggered release. Thus, synaptotagmin-2 is an essential synaptotagmin isoform that functions in concert with other synaptotagmins in the Ca2+ triggering of neurotransmitter release.
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Encéfalo/metabolismo , Calcio/fisiología , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Sinapsis/metabolismo , Sinaptotagmina II/fisiología , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Neuromuscular/genética , Neurotransmisores/genética , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Sinapsis/genética , Transmisión Sináptica/genética , Sinaptotagmina II/deficiencia , Sinaptotagmina II/genéticaRESUMEN
The mammalian muscle nicotinic acetylcholine receptor (AChR) is composed of five membrane-spanning subunits and its composition differs between embryonic and adult muscles. In embryonic muscles, it is composed of two alpha-, one beta-, one delta-, and one gamma-subunit; the gamma-subunit is later replaced by the epsilon-subunit during postnatal development. This unique temporal expression pattern of the gamma-subunit suggests it may play specific roles in embryonic muscles. To address this issue, we examined the formation and function of the neuromuscular junction in mouse embryos deficient in the gamma-subunit. At embryonic day 15.5, AChR clusters were absent and the spontaneous miniature endplate potentials were undetectable in the mutant muscles. However, electrical stimulation of the nerves triggered muscle contraction and elicited postsynaptic endplate potential (EPP) in the mutant muscles, although the magnitude of the muscle contraction and the amplitudes of the EPPs were smaller in the mutant compared to the wild-type muscles. Reintroducing a wild-type gamma-subunit into the mutant myotubes restored the formation of AChR clusters in vitro. Together, these results have demonstrated that functional AChRs were present in the mutant muscle membrane, but at reduced levels. Thus, in the absence of the gamma-subunit, a combination of alpha, beta, and delta subunits may assemble into functional receptors in vivo. These results also suggest that the gamma-subunit maybe involved in interacting with rapsyn, a cytoplasmic protein required for AChR clustering.
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Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/metabolismo , Subunidades de Proteína/genética , Agregación de Receptores/genética , Receptores Nicotínicos/metabolismo , Animales , Células Cultivadas , Diafragma/crecimiento & desarrollo , Diafragma/inervación , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/genética , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/fisiología , Proteínas Musculares/metabolismo , Unión Neuromuscular/genética , Técnicas de Cultivo de Órganos , Nervio Frénico/crecimiento & desarrollo , Receptores Nicotínicos/genética , Potenciales Sinápticos/genéticaRESUMEN
Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre- and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR gamma-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the gamma-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in gamma-null muscles. These results demonstrate that the gamma-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.
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Unión Neuromuscular/metabolismo , Organogénesis/fisiología , Receptores Colinérgicos/fisiología , Acetilcolinesterasa/metabolismo , Animales , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Electrofisiología , Inmunohistoquímica , Ratones , Ratones Mutantes , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología , Proteínas Musculares/metabolismo , Músculos/embriología , Músculos/metabolismo , Unión Neuromuscular/embriología , Unión Neuromuscular/genética , Organogénesis/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Alpha-RIMs (RIM1alpha and RIM2alpha) are multidomain active zone proteins of presynaptic terminals. Alpha-RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N-terminal region, and interact with other synaptic proteins via their central and C-terminal regions. Although RIM1alpha has been well characterized, nothing is known about the function of RIM2alpha. We now show that RIM1alpha and RIM2alpha are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2alpha, and crossed them with previously produced RIM1alpha knockout mice. We found that deletion of either RIM1alpha or RIM2alpha is not lethal, but ablation of both alpha-RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without alpha-RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca(2+)-triggered release. Our data thus demonstrate that alpha-RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca(2+)-triggering of exocytosis.