Peripheral Synapses Research Articles

GFP and FAP imaging of neuropeptide release in Drosophila.

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca2+ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP.

Multi targets of cannabidiol (CBD) on skeletal mammalian and avian neuromuscular preparations

Cannabidiol (CBD) has been used in diseases that affect the central nervous system. Its effects on the peripheral synapses are of great interest, since endocannabinoid receptors are expressed in muscles. CBD (0.3 mM) was analysed using mammalian and avian neuromuscular preparations, through myographic techniques in complementary protocols. Mammalian cells were examined by light microscopy while exogenous acetylcholine (40 µM) and potassium chloride (100 mM) were added into avian preparations, before and at the end of experiments. Pharmacological tools such as atropine (2 µM), polyethylene glycol (PEG 400, 20 µM), Ca2+ (1.8 mM), F55-6 (20 µg/mL), and nifedipine (1.3 mM) were assessed with CBD. In mice, CBD causes a facilitatory effect and paralysis, whereas in avian, paralysis. Concluding, CBD is responsible for activated or inhibited channels, for ACh release via muscarinic receptor modulation, and by the inhibition of nicotinic receptors leading to neuromuscular blockade, with no damage to striated muscle cells.

Oxysterols in Central and Peripheral Synaptic Communication.

Cholesterol is a key molecule for synaptic transmission, and both central and peripheral synapses are cholesterol rich. During intense neuronal activity, a substantial portion of synaptic cholesterol can be oxidized by either enzymatic or non-enzymatic pathways to form oxysterols, which in turn modulate the activities of neurotransmitter receptors (e.g., NMDA and adrenergic receptors), signaling molecules (nitric oxide synthases, protein kinase C, liver X receptors), and synaptic vesicle cycling involved in neurotransmitters release. 24-Hydroxycholesterol, produced by neurons in the brain, could directly affect neighboring synapses and change neurotransmission. 27-Hydroxycholesterol, which can cross the blood-brain barrier, can alter both synaptogenesis and synaptic plasticity. Increased generation of 25-hydroxycholesterol by activated microglia and macrophages could link inflammatory processes to learning and neuronal regulation. Amyloids and oxidative stress can lead to an increase in the levels of ring-oxidized sterols and some of these oxysterols (4-cholesten-3-one, 5α-cholestan-3-one, 7β-hydroxycholesterol, 7-ketocholesterol) have a high potency to disturb or modulate neurotransmission at both the presynaptic and postsynaptic levels. Overall, oxysterols could be used as "molecular prototypes" for therapeutic approaches. Analogs of 24-hydroxycholesterol (SGE-301, SGE-550, SAGE718) can be used for correction of NMDA receptor hypofunction-related states, whereas inhibitors of cholesterol 24-hydroxylase, cholestane-3β,5α,6β-triol, and cholest-4-en-3-one oxime (olesoxime) can be utilized as potential anti-epileptic drugs and (or) protectors from excitotoxicity.

The Axon Initial Segment Plays a Dynamic Role in Peripheral Motor Neuron Synapse Regeneration following Injury.

Unlike axons of the central nervous system, which have limited ability to regenerate, axons of the peripheral nervous system (PNS) die back and can then reform in response to injury, through a process called Wallerian degeneration ([Huebner and Strittmatter, 2009][1]). However, severe peripheral

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12h/day for 5 or 8days. NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P<0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7±0.5 and 6.9±0.5 sprouts per NMJ, respectively, compared with 10.3±0.9 tSC per NMJ (P<0.001) in non-stimulated soleus from rats immobilized for 8days. A similar reduction of axonal sprouts (P<0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

A method to identify, dissect and stain equine neuromuscular junctions for morphological analysis.

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot–Marie–Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross‐species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole‐mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.

NMDAR-dependent presynaptic homeostasis in adult hippocampus: Synapse growth and cross-modal inhibitory plasticity

Homeostatic plasticity (HP) encompasses a suite of compensatory physiological processes that counteract neuronal perturbations, enabling brain resilience. Currently, we lack a complete description of the homeostatic processes that operate within the mammalian brain. Here, we demonstrate that acute, partial AMPAR-specific antagonism induces potentiation of presynaptic neurotransmitter release in adult hippocampus, a form of compensatory plasticity that is consistent with the expression of presynaptic homeostatic plasticity (PHP) documented at peripheral synapses. We show that this compensatory plasticity can be induced within minutes, requires postsynaptic NMDARs, and is expressed via correlated increases in dendritic spine volume, active zone area, and docked vesicle number. Further, simultaneous postsynaptic genetic reduction of GluA1, GluA2, and GluA3 in triple heterozygous knockouts induces potentiation of presynaptic release. Finally, induction of compensatory plasticity at excitatory synapses induces a parallel, NMDAR-dependent potentiation of inhibitory transmission, a cross-modal effect consistent with the anti-epileptic activity of AMPAR-specific antagonists used in humans.

Abstract 057: Macula Densa Stimulation Acutely Increases Afferent Renal Nerve Activity And Blood Pressure

Macula densa (MD) cells in the kidney are known to regulate renal hemodynamics and the renin-angiotensin system, and indirectly systemic blood pressure (BP). However, neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the hypothesis that MD cells function similarly to peripheral ganglia, form neuronal networks that are part of the peripheral (renal) nervous system and synapse with renal afferent/efferent nerves as a new pathway for the direct control of sympathetic tone and BP. Single cell MD Ca 2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-G6 mice that selectively express the ratiometric Ca 2+ reporter GCaMP6f/tdTomato in MD cells. Afferent renal nerve activity (ARNA) was isolated and measured via recording electrodes. Systemic BP was monitored via the cannulated carotid artery. Optogenetic MD stimulation was induced by blue light (470nm) exposure of the whole left kidney of MD-Ai27 mice that selectively express the depolarizing channelrhodopsin ChR2 in MD cells. Pacemaker-like regular Ca 2+ oscillations (4-fold elevations in baseline Ca 2+ and frequency of 0.03 Hz) and their cell-to-cell propagation via long, axon-like processes were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high expression of genes related to synaptic transmission that was validated by immunohistochemistry. Acute intra-renal infusion of arginine-vasopressin (AVP) produced 3-fold increase in MD Ca 2+ firing frequency (via the MD-specific AVPR1a) and a dose-dependent increase in ARNA and BP (saline: -1±1; 0.01ug/mL: 2±1; 0.1ug/mL: 7±2; 0.25ug/mL: 7±3; 0.5ug/mL: 10±3; 1.0ug/mL: 23±13). IV administration of AVP (0.25ug/mL) did not significantly alter ARNA (108±3%, n=3) but produced a significant increase in arterial blood pressure (40±6mmHg, n=3). Blue light exposure of MD-Ai27 mouse kidneys acutely and reversibly increased systemic BP by 22±5mmHg (P&lt;0.05, n=5). In summary, MD cells send interoceptive signals to the brain via renal sensory afferent nerves to directly control BP. Targeting of novel MD mechanisms may result in exciting new therapeutic opportunities for renal and cardiovascular diseases and hypertension.

Butyrylcholinesterase distribution in the mouse gastrointestinal tract: An immunohistochemical study.

Butyrylcholinesterase (BChE) is a hydrolytic enzyme that together with acetylcholinesterase (AChE) belongs to the cholinesterase family. Whereas AChE has a well-established role in regulating cholinergic neurotransmission in central and peripheral synapses, the physiological role of BChE remains elusive. In this morphological immunohistochemical and double-label confocal microscopy study we investigated the distribution of BChE in the mouse gastrointestinal tract. BChE-positive cells were detected in the liver (both in hepatocytes and cholangiocytes), in the keratinised layers of the squamous epithelium of the oesophagus and forestomach, in the oxyntic mucosa of the stomach, in the mucus-secreting cells of duodenal Brunner glands and the small and large intestinal mucosa. Interestingly, BChE-positive cells were often detected close to gastrointestinal proliferative niches. In the oxyntic mucosa, the close proximity of ghrelin-producing and BChE-positive parietal cells suggests that BChE may be involved in ghrelin hydrolysation through paracrine action. To our knowledge, this is the first comprehensive morphological study performed to gain insight into the physiological role of BChE in the gastrointestinal tract.

Functional regeneration of the murine neuromuscular synapse relies on long-lasting morphological adaptations

BackgroundIn a broad variety of species, muscle contraction is controlled at the neuromuscular junction (NMJ), the peripheral synapse composed of a motor nerve terminal, a muscle specialization, and non-myelinating terminal Schwann cells. While peripheral nerve damage leads to successful NMJ reinnervation in animal models, muscle fiber reinnervation in human patients is largely inefficient. Interestingly, some hallmarks of NMJ denervation and early reinnervation in murine species, such as fragmentation and poly-innervation, are also phenotypes of aged NMJs or even of unaltered conditions in other species, including humans. We have reasoned that rather than features of NMJ decline, such cellular responses could represent synaptic adaptations to accomplish proper functional recovery. Here, we have experimentally tackled this idea through a detailed comparative study of the short- and long-term consequences of irreversible (chronic) and reversible (partial) NMJ denervation in the convenient cranial levator auris longus muscle.ResultsOur findings reveal that irreversible muscle denervation results in highly fragmented postsynaptic domains and marked ectopic acetylcholine receptor clustering along with significant terminal Schwann cells sprouting and progressive detachment from the NMJ. Remarkably, even though reversible nerve damage led to complete reinnervation after 11 days, we found that more than 30% of NMJs are poly-innervated and around 65% of postsynaptic domains are fragmented even 3 months after injury, whereas synaptic transmission is fully recovered two months after nerve injury. While postsynaptic stability was irreversibly decreased after chronic denervation, this parameter was only transiently affected by partial NMJ denervation. In addition, we found that a combination of morphometric analyses and postsynaptic stability determinations allows discriminating two distinct forms of NMJ fragmentation, stable-smooth and unstable-blurred, which correlate with their regeneration potential.ConclusionsTogether, our data unveil that reversible nerve damage imprints a long-lasting reminiscence in the NMJ that results in the rearrangement of its cellular components. Instead of being predictive of NMJ decline, these traits may represent an efficient adaptive response for proper functional recovery. As such, these features are relevant targets to be considered in strategies aimed to restore motor function in detrimental conditions for peripheral innervation.

Effects of cannabinoid and vanilloid receptor antagonists on nicotine induced relaxation response enhancement in rabbit corpus cavernosum.

Objective(s):Endocannabinoids and nicotine regulate the neurotransmitter release in different central and peripheral synapses. Various studies in the literature demonstrate the interaction between endocannabinoid and nicotinic systems, especially in the central nervous system. The interaction between nicotinic and endocannabinoid systems was investigated in this study. We aimed to show the effects of cannabinoid and vanilloid receptor antagonists on nicotine-induced relaxation response increases in rabbit corpus cavernosum.Materials and Methods:From a total of seven male albino rabbits, three or four equal strips were cut from each corpus cavernosum and inserted in isolated organ baths. Tissues were contracted with phenylephrine (3×10−5 M). After contraction reached a plateau, strips were stimulated with EFS, and with the stabilization of EFS relaxation responses, 10-4 M of nicotine was administered to tissues. After that, in order to investigate the effects of AM251 (CB1 antagonist), AM630 (CB2 inverse agonist) or capsazepine (a vanilloid receptor antagonist) were given to different tissues, after the resting period.Results:Nicotine (10−4 M) increased the EFS-induced relaxation responses (14.60%±2.94%, P<0.05). AM630 decreased the enhancement of nicotine-induced EFS relaxation responses (nicotine 10-4 M enhancement: 17.16%±3.19%; nicotine 10-4 M enhancement in the presence of AM630 10-6 M: 4.44%±3.43% P<0.05; n=6), whereas effects of AM251 and capsazepine were not significant. Conclusion:In the present study, nicotine increased the amplitudes of EFS-induced relaxation responses probably via nicotinic acetylcholine receptors located on the nitrergic nerves of the corpus cavernosum. We showed the role of cannabinoid-like endo-ligands in nicotine-induced enhancement via CB2 receptors but not CB1 and VR1 receptors.

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

Maturation of a postsynaptic domain: Role of small Rho GTPases in organising nicotinic acetylcholine receptor aggregates at the vertebrate neuromuscular junction.

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR-containing plaques to elaborate and branched pretzel-like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome-like structures to define discrete low nAChR-containing regions to consolidate a plaque-to-pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome-like structures that drive the postsynaptic maturation of vertebrate NMJs.

Noncanonical Activity of Endocannabinoids and Their Receptors in Central and Peripheral Synapses.

This review focuses on new aspects of endocannabinoid functions and mechanisms of activity in central and peripheral synapses, different from the general viewpoint that endocannabinoids are retrograde signaling molecules, which inhibit neurotransmitter release by activating specific presynaptic endocannabinoid receptors CB1 and CB2. Biased agonism of the endogenous and synthetic cannabinoids as well as ability of the CB-receptors to couple not only with classical Gi-proteins, but also with Gs- and Gq-proteins and, moreover, with β-arrestins (thereby triggering additional signaling pathways in synapses) are described here in detail. Examples of noncanonical tonic activity of endocannabinoids and their receptors and their role in synaptic function are also presented. The role of endocannabinoids in short-term and long-term potentiation of neurotransmitter release in central synapses and their facilitating effect on quantal size and other parameters of acetylcholine release in mammalian neuromuscular junctions are highlighted in this review. In conclusion, it is stated that the endocannabinoid system has a wider range of various multidirectional modulating effects (both potentiating and inhibiting) on neurotransmitter release than initially recognized. Re-evaluation of the functions of endocannabinoid system with consideration of its noncanonical features will lead to better understanding of its role in the normal and pathological functioning of the nervous system and other systems of the body, which has an enormous practical value.

NMJ-Analyser identifies subtle early changes in mouse models of neuromuscular disease

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fibre. NMJs are thought to be the primary site of peripheral pathology in many neuromuscular diseases, but innervation/denervation status is often assessed qualitatively with poor systematic criteria across studies, and separately from 3D morphological structure. Here, we describe the development of ‘NMJ-Analyser’, to comprehensively screen the morphology of NMJs and their corresponding innervation status automatically. NMJ-Analyser generates 29 biologically relevant features to quantitatively define healthy and aberrant neuromuscular synapses and applies machine learning to diagnose NMJ degeneration. We validated this framework in longitudinal analyses of wildtype mice, as well as in four different neuromuscular disease models: three for amyotrophic lateral sclerosis (ALS) and one for peripheral neuropathy. We showed that structural changes at the NMJ initially occur in the nerve terminal of mutant TDP43 and FUS ALS models. Using a machine learning algorithm, healthy and aberrant neuromuscular synapses are identified with 95% accuracy, with 88% sensitivity and 97% specificity. Our results validate NMJ-Analyser as a robust platform for systematic and structural screening of NMJs, and pave the way for transferrable, and cross-comparison and high-throughput studies in neuromuscular diseases.

Synapse-specific Lrp4 mRNA enrichment requires Lrp4/MuSK signaling, muscle activity and Wnt non-canonical pathway

BackgroundThe neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed.ResultsWe studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression.ConclusionsThis study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse.

ABSTRACTThe coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non‐myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.

Chronic Pharmacological Increase of Neuronal Activity Improves Sensory-Motor Dysfunction in Spinal Muscular Atrophy Mice.

Dysfunction of neuronal circuits is an important determinant of neurodegenerative diseases. Synaptic dysfunction, death, and intrinsic activity of neurons are thought to contribute to the demise of normal behavior in the disease state. However, the interplay between these major pathogenic events during disease progression is poorly understood. Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the ubiquitously expressed protein SMN and is characterized by motor neuron death, skeletal muscle atrophy, as well as dysfunction and loss of both central and peripheral excitatory synapses. These disease hallmarks result in an overall reduction of neuronal activity in the spinal sensory-motor circuit. Here, we show that increasing neuronal activity by chronic treatment with the FDA-approved potassium channel blocker 4-aminopyridine (4-AP) improves motor behavior in both sexes of a severe mouse model of SMA. 4-AP restores neurotransmission and number of proprioceptive synapses and neuromuscular junctions (NMJs), while having no effects on motor neuron death. In addition, 4-AP treatment with pharmacological inhibition of p53-dependent motor neuron death results in additive effects, leading to full correction of sensory-motor circuit pathology and enhanced phenotypic benefit in SMA mice. Our in vivo study reveals that 4-AP-induced increase of neuronal activity restores synaptic connectivity and function in the sensory-motor circuit to improve the SMA motor phenotype.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is a neurodegenerative disease, characterized by synaptic loss, motor neuron death, and reduced neuronal activity in spinal sensory-motor circuits. However, whether these are parallel or dependent events is unclear. We show here that long-term increase of neuronal activity by the FDA-approved drug 4-aminopyridine (4-AP) rescues the number and function of central and peripheral synapses in a SMA mouse model, resulting in an improvement of the sensory-motor circuit and motor behavior. Combinatorial treatment of pharmacological inhibition of p53, which is responsible for motor neuron death and 4-AP, results in additive beneficial effects on the sensory-motor circuit in SMA. Thus, neuronal activity restores synaptic connections and improves significantly the severe SMA phenotype.