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BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

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Epilepsy is a neurological disorder characterized by a hyperexcitable state in neurons from different brain regions. Much is unknown about epilepsy and seizures development, depicting a growing field of research. Animal models have provided important clues about the underlying mechanisms of seizure-generating neuronal circuits. Mammalian complexity still makes it difficult to define some principles of nervous system function, and non-mammalian models have played pivotal roles depending on the research question at hand. Mollusks and the Helix land snail have been used to study epileptic-like behavior in neurons. Neurons from these organisms confer advantages as single-cell identification, isolation, and culture, either as single cells or as physiological relevant monosynaptic or polysynaptic circuits, together with amenability to different protocols and treatments. This review's purpose consists in presenting relevant papers in order to gain a better understanding of Helix neurons, their characteristics, uses, and capabilities for studying the fundamental mechanisms of epileptic disorders and their treatment, to facilitate their more expansive use in epilepsy research.

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Two types of secretory vesicles co-exist at some presynaptic terminals. Clear synaptic vesicles (CSV) release their contents at the synaptic active zone, upon single impulses, while dense-core vesicles (DCV) usually release their contents in the periphery of the terminal upon repetitive stimulation. Part of the transmitter released by DCV diffuses to produce paracrine effects, and part of it reaches the postsynaptic terminal, adding its effect to that of synaptic release. This article presents an analytical method to separate the contribution of CSV and DCV to the postsynaptic responses, based on the kinetics of postsynaptic currents (PSCs). Since stimulation with single presynaptic impulses usually triggers release only from CSV, the kinetics of the resulting PSC can be used as a template to model the postsynaptic response to release from CSV during stimulation trains, accounting for the variations in the amplitude of PSCs due to short-term synaptic plasticity. Subtraction of this model simulation to the total recorded PSC renders the response to DCV peri­synaptic release, which has slower kinetics. The method can be further simplified by measuring only the amplitudes of the PSC peaks for synaptic release and the integral of the current for peri­synaptic release.•The postsynaptic current in response to presynaptic release from clear synaptic vesicles is modeled using the kinetics of the PSC in response to single impulses.•The model synaptic response is subtracted from the total recorded PSC to obtain the response to peri­synaptic release from dense-core vesicles.

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The coordinated movement of organisms relies on efficient nerve-muscle communication at the neuromuscular junction. After peripheral nerve injury or neurodegeneration, motor neurons and Schwann cells increase the expression of the p75NTR pan-neurotrophin receptor. Even though p75NTR targeting has emerged as a promising therapeutic strategy to delay peripheral neuronal damage progression, the effects of long-term p75NTR inhibition at the mature neuromuscular junction have not been elucidated. We performed quantitative neuroanathomical analyses of the neuromuscular junction in p75NTR null mice by laser confocal and electron microscopy, which were complemented with electromyography, locomotor tests, and pharmacological intervention studies. Mature neuromuscular synapses of p75NTR null mice show impaired postsynaptic organization and ultrastructural complexity, which correlate with altered synaptic function at the levels of nerve activity-induced muscle responses, muscle fiber structure, force production, and locomotor performance. Our results on primary myotubes and denervated muscles indicate that muscle-derived p75NTR does not play a major role on postsynaptic organization. In turn, motor axon terminals of p75NTR null mice display a strong reduction in the number of synaptic vesicles and active zones. According to the observed pre and postsynaptic defects, pharmacological acetylcholinesterase inhibition rescued nerve-dependent muscle response and force production in p75NTR null mice. Our findings revealing that p75NTR is required to organize mature neuromuscular junctions contribute to a comprehensive view of the possible effects caused by therapeutic attempts to target p75NTR.

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Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

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Huntington's disease (HD) is a neurodegenerative disorder characterized by a progressive decline of motor and cognitive functions. It is caused by a polyglutamine expansion in the huntingtin (htt) protein, which then leads to neurodegeneration that span both the central and peripheral nervous system. Previous works have shown that htt interacts with several proteins from the neurotransmitter release machinery causing synaptic dysfunction. In this work, we looked for alterations in diaphragm neuromuscular junctions (NMJs) from 3 to 4 months old BACHD mouse model for HD. This model represents a new and robust in vivo paradigm for studying the pathogenesis of HD. For optical analysis, NMJs were stained with FM1-43fx and α-bungarotoxin to visualize both pre and postsynaptic elements, respectively. Confocal microscopy optical analysis showed a decrease in the number of synaptic elements and fluorescence intensity in NMJs from BACHD diaphragms compared to WT. We next analyzed presynaptic activity and we observed that synaptic vesicle exocytosis was impaired in NMJs from BACHD diaphragms. Ultrastructural analysis revealed significant changes in the form and sizes of the synaptic vesicles in BACHD diaphragm NMJs that could contribute to impaired exocytosis. Additionally, electrophysiology recordings revealed a decrease in the amplitude of miniature endplate potentials (MEPPs) from BACHD diaphragm NMJs. Our data suggest a dysfunction in BACHD diaphragm NMJs that might occur in other muscles and may aggravate the motor defects seen in HD. These results may contribute to a better understanding of peripheral cholinergic dysfunction in this neurodegenerative disease.

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Ganglionic synapses have the capability to express long-term potentiation (gLTP) after application of a brief high-frequency stimulus. It has been suggested a possible role of gLTP in some cardiovascular diseases. Although a number of characteristics of gLTP have been described, the precise locations and mechanisms underlying gLTP are not completely known. Current findings support two major conflicting presynaptic and postsynaptic hypotheses. The presynaptic hypothesis posits a presynaptic increase in acetylcholine (ACh) release, whereas the postsynaptic hypothesis proposes a long-lasting enhancement of the nicotinic response on the postsynaptic membrane. An alternative trans-synaptic hypothesis proposes the presynaptic release of a cotransmitter from large dense core vesicles, which postsynaptically enhances synaptic efficacy and accounts for gLTP. Here, we review the studies of LTP, with emphasis on gLTP in mammals, and we examine the findings that support the presynaptic, the postsynaptic and the trans-synaptic hypotheses. We then review our data on the contribution of calcium to gLTP as an approach to elucidate the mechanisms of gLTP. Data on the contribution of calcium to gLTP and on prolonged high-frequency stimulus-dependent fading of LTP have led us to support the trans-synaptic process as responsible for gLTP. Finally, we present a formal working model for the mechanisms of gLTP.

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