Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle.
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ABSTRACT:
SUBMITTER: Brunelli G
PROVIDER: S-EPMC1142481 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. The onset of PNI is characterized by nerve degeneration distal to the nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generated a mouse model in which NANOG, a pluripotency-associated transcription factor can be expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulated the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression led to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation, and downregulation of key muscle atrophy genes. Further, NANOG mice demonstrated extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice showed greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming the muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.
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Project description:BackgroundPeripheral nerve injury is a frequent cause of lasting motor deficits and chronic pain. Although peripheral nerves are capable of regrowth they often fail to re-innervate target tissues.ResultsUsing newly generated transgenic mice with inducible neuronal progranulin overexpression we show that progranulin accelerates axonal regrowth, restoration of neuromuscular synapses and recovery of sensory and motor functions after injury of the sciatic nerve. Oppositely, progranulin deficient mice have long-lasting deficits in motor function tests after nerve injury due to enhanced losses of motor neurons and stronger microglia activation in the ventral horn of the spinal cord. Deep proteome and gene ontology (GO) enrichment analysis revealed that the proteins upregulated in progranulin overexpressing mice were involved in 'regulation of transcription' and 'response to insulin' (GO terms). Transcription factor prediction pointed to activation of Notch signaling and indeed, co-immunoprecipitation studies revealed that progranulin bound to the extracellular domain of Notch receptors, and this was functionally associated with higher expression of Notch target genes in the dorsal root ganglia of transgenic mice with neuronal progranulin overexpression. Functionally, these transgenic mice recovered normal gait and running, which was not achieved by controls and was stronger impaired in progranulin deficient mice.ConclusionWe infer that progranulin activates Notch signaling pathways, enhancing thereby the regenerative capacity of partially injured neurons, which leads to improved motor function recovery.
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Project description:We assessed synaptic ?-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) properties during synaptogenesis to describe the development of individual glutamatergic synapses on rat hippocampal CA1 principal neurons. Pharmacologically isolated AMPAR-mediated glutamatergic synaptic currents [evoked by stimulation of the Schaffer Collateral pathway, excitatory postsynaptic currents (EPSCs)], had significantly greater inward-rectification at ages P5-7 compared with P8-18. These inward rectifying EPSCs demonstrated paired-pulse dependent unblocking at positive holding potentials, consistent with voltage-dependent internal polyamine block. Measurements of paired-pulse facilitation did not support altered presynaptic properties associated with inward rectification. Using asynchronous EPSCs (aEPSCs) to analyze populations of individual synapses, we found that quantal amplitudes (Q) increased across early postnatal development (P5-P18) and were directly modulated by increases in the number of activated receptors. Quantal AMPAR decay kinetics (aEPSC ?(decay)s) exhibited the highest coefficient of variation (CV) from P5 to 7 and became markedly less variable at P8-18. At P5-7, faster quantal kinetics coexisted with much slower kinetics; only slower quantal kinetics were found at P8-18. This supports diverse quantal synaptic properties limited to P5-7. Multivariate cluster analysis of Q, CV(? decay), and median ?(decay) supported a segregation of neurons into two distinct age groups of P5-7 and P8-18, similar to the age-related segregation suggested by inward rectification. Taken together, these findings support synaptic, calcium permeable AMPARs at a subset of synapses onto CA1 pyramidal neurons exclusively at P5-7. These distinct synapses coexist with those sharing the properties of more mature synapses. These synapses disappear after P7 as activated receptor numbers increase with age.
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Project description:Synaptic excitation mediates a broad spectrum of structural changes in neural circuits across the brain. Here, we examine the morphologies, wiring, and architectures of single synapses of projection neurons in the murine hippocampus that developed in virtually complete absence of vesicular glutamate release. While these neurons had smaller dendritic trees and/or formed fewer contacts in specific hippocampal subfields, their stereotyped connectivity was largely preserved. Furthermore, loss of release did not disrupt the morphogenesis of presynaptic terminals and dendritic spines, suggesting that glutamatergic neurotransmission is unnecessary for synapse assembly and maintenance. These results underscore the instructive role of intrinsic mechanisms in synapse formation.
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Project description:Three male 8-week-old Wistar rats were used. The peroneal nerve of the animals was transected and sutured to the biceps femoris muscle. Four weeks later, the proximal stump was sutured to the distal stump. The animals survived for 8 weeks. All experiments were performed under Ketanest (ketamine hydrochloride, Essex Pharma GmbH, Munich, Germany; 100 mg/kg) and Rompun (xylazine hydrochloride, Bayer, Leverkusen, Germany; 10 mg/kg) anesthesia. Both substances were injected intraperitoneally. RNA was extracted from the ipsilateral and contralateral tibialis anterior muscles of each animal. The gene array analysis compared the ipsilateral muscle with the contralateral muscle of each animal. Keywords: parallel sample
2005-04-28 | GSE2566 | GEO
Project description:Optimal function of neuronal networks requires interplay between rapid forms of Hebbian plasticity and homeostatic mechanisms that adjust the threshold for plasticity, termed metaplasticity. Numerous forms of rapid synapse plasticity have been examined in detail. However, the rules that govern synaptic metaplasticity are much less clear. Here, we demonstrate a local subunit-specific switch in NMDA receptors that alternately primes or prevents potentiation at single synapses. Prolonged suppression of neurotransmitter release enhances NMDA receptor currents, increases the number of functional NMDA receptors containing NR2B, and augments calcium transients at single dendritic spines. This local switch in NMDA receptors requires spontaneous glutamate release but is independent of action potentials. Moreover, single inactivated synapses exhibit a lower induction threshold for both long-term synaptic potentiation and plasticity-induced spine growth. Thus, spontaneous glutamate release adjusts plasticity threshold at single synapses by local regulation of NMDA receptors, providing a novel spatially delimited form of synaptic metaplasticity.
| S-EPMC2911980 | biostudies-literature
Project description:Glutamate (Glu) is the primary excitatory transmitter in the mammalian brain. But, we know little about the evolutionary history of this adaptation, including the selection of l-glutamate as a signaling molecule in the first place. Here, we used comparative metabolomics and genomic data to reconstruct the genealogy of glutamatergic signaling. The origin of Glu-mediated communications might be traced to primordial nitrogen and carbon metabolic pathways. The versatile chemistry of L-Glu placed this molecule at the crossroad of cellular biochemistry as one of the most abundant metabolites. From there, innovations multiplied. Many stress factors or injuries could increase extracellular glutamate concentration, which led to the development of modular molecular systems for its rapid sensing in bacteria and archaea. More than 20 evolutionarily distinct families of ionotropic glutamate receptors (iGluRs) have been identified in eukaryotes. The domain compositions of iGluRs correlate with the origins of multicellularity in eukaryotes. Although L-Glu was recruited as a neuro-muscular transmitter in the early-branching metazoans, it was predominantly a non-neuronal messenger, with a possibility that glutamatergic synapses evolved more than once. Furthermore, the molecular secretory complexity of glutamatergic synapses in invertebrates (e.g., Aplysia) can exceed their vertebrate counterparts. Comparative genomics also revealed 15+ subfamilies of iGluRs across Metazoa. However, most of this ancestral diversity had been lost in the vertebrate lineage, preserving AMPA, Kainate, Delta, and NMDA receptors. The widespread expansion of glutamate synapses in the cortical areas might be associated with the enhanced metabolic demands of the complex brain and compartmentalization of Glu signaling within modular neuronal ensembles.
| S-EPMC9233959 | biostudies-literature
Project description:BackgroundThe functioning of the nervous system depends upon the specificity of its synaptic contacts. The mechanisms triggering the expression of the appropriate receptors on postsynaptic membrane and the role of the presynaptic partner in the differentiation of postsynaptic structures are little known.Methods and findingsTo address these questions we cocultured murine primary muscle cells with several glutamatergic neurons, either cortical, cerebellar or hippocampal. Immunofluorescence and electrophysiology analyses revealed that functional excitatory synaptic contacts were formed between glutamatergic neurons and muscle cells. Moreover, immunoprecipitation and immunofluorescence experiments showed that typical anchoring proteins of central excitatory synapses coimmunoprecipitate and colocalize with rapsyn, the acetylcholine receptor anchoring protein at the neuromuscular junction.ConclusionsThese results support an important role of the presynaptic partner in the induction and differentiation of the postsynaptic structures.
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Project description:Peripheral nerves have a propensity for axon growth and regeneration that the central nervous system lacks (CNS). However, CNS axons can also grow long distances if introduced to a graft harvested from a peripheral nerve (PNGs), which is the rationale for using PNGs as repair strategy for injuries of the spinal cord. From a clinical perspective, PNGs provide interesting possibilities with potential to repair the injured spinal cord. First, there are numerous options to harvest autologous grafts associated with low risk for the patient. Second, a PNG allow axons to grow considerable distances and can, by the surgical procedure, be navigated to specific target sites in the CNS. Furthermore, a PNG provides all necessary biological substrates for myelination of elongating axons. A PNG can thus be suited to bridge axons long distances across an injury site and restore long tracts in incomplete SCI. Experimentally, locomotor functions have been improved transplanting a PNG after incomplete injury. However, we still know little with regard to the formation of new circuitries and functional outcome in association to when, where, and how grafts are inserted into the injured spinal cord, especially for sensory functions. In this perspective, we discuss the advantages of PNG from a clinical and surgical perspective, the need for adding/repairing long tracts, how PNGs are best applied for incomplete injuries, and the unexplored areas we believe are in need of answers.
| S-EPMC9097274 | biostudies-literature
Project description:Skeletal muscle-derived cells have strong secretory function, while skeletal muscle-derived stem cells, which are included in muscle-derived cells, can differentiate into Schwann cell-like cells and other cell types. However, the effect of muscle-derived cells on peripheral nerve defects has not been reported. In this study, 5-mm-long nerve defects were created in the right sciatic nerves of mice to construct a peripheral nerve defect model. Adult female C57BL/6 mice were randomly divided into four groups. For the muscle-derived cell group, muscle-derived cells were injected into the catheter after the cut nerve ends were bridged with a polyurethane catheter. For external oblique muscle-fabricated nerve conduit and polyurethane groups, an external oblique muscle-fabricated nerve conduit or polyurethane catheter was used to bridge the cut nerve ends, respectively. For the sham group, the sciatic nerves on the right side were separated but not excised. At 8 and 12 weeks post-surgery, distributions of axons and myelin sheaths were observed, and the nerve diameter was calculated using immunofluorescence staining. The number, diameter, and thickness of myelinated nerve fibers were detected by toluidine blue staining and transmission electron microscopy. Muscle fiber area ratios were calculated by Masson's trichrome staining of gastrocnemius muscle sections. Sciatic functional index was recorded using walking footprint analysis at 4, 8, and 12 weeks after operation. The results showed that, at 8 and 12 weeks after surgery, myelin sheaths and axons of regenerating nerves were evenly distributed in the muscle-derived cell group. The number, diameter, and myelin sheath thickness of myelinated nerve fibers, as well as gastrocnemius muscle wet weight and muscle area ratio, were significantly higher in the muscle-derived cell group compared with the polyurethane group. At 4, 8, and 12 weeks post-surgery, sciatic functional index was notably increased in the muscle-derived cell group compared with the polyurethane group. These criteria of the muscle-derived cell group were not significantly different from the external oblique muscle-fabricated nerve conduit group. Collectively, these data suggest that muscle-derived cells effectively accelerated peripheral nerve regeneration. This study was approved by the Animal Ethics Committee of Plastic Surgery Hospital, Chinese Academy of Medical Sciences (approval No. 040) on September 28, 2016.
| S-EPMC6862419 | biostudies-literature