Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle

Abstract

Acetylcholine is the main neurotransmitter at the mammalian neuromuscular junction (NMJ) where nicotinic acetylcholine receptors mediate the signaling between nerve terminals and muscle fibers. We show that under glutamatergic transmission, rat NMJ switches from cholinergic type synapse to glutamatergic synapse. Connecting skeletal muscle to the lateral white matter of the spinal cord by grafting the distal stump of the transected motor nerve produced functional muscle reinnervation. The restored neuromuscular activity became resistant to common curare blockers but sensitive to the glutamate ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Analysis of the regenerated nerve disclosed new glutamatergic axons and the disappearance of cholinergic fibers. Many axons belonged to the supraspinal neurons located in the red nucleus and the brainstem nuclei. Finally, the innervated muscle displayed high expression and clustering of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits glutamate receptors 1 and 2. Our data suggest that supraspinal neurons can target skeletal muscle, which retains the plasticity to generate functional glutamatergic NMJ.glutamate ͉ neuromuscular junction ͉ red nucleus T raumatic paraplegia caused by spinal cord injury is still an irreversible condition. So far, there is no medical or surgical treatment capable of curing paraplegia. The CNS is ''nonpermissive'' for the advancement of injured axons because of an abundance of growth-inhibitory molecules in the myelin and the glial scar (1-3). In addition, there are no growth-promoting factors at the neuronal growth cone or at the somata. However, when peripheral nerves (PN) are directly grafted into the CNS, central axons can progress throughout the peripheral endoneural tubes, suggesting they can regenerate in an appropriate environment (4-7). The central fibers that are diverted into a nerve graft implanted within a healthy structure derive from neurons axotomized during the grafting procedure and not from uninjured neurons spared by nerve graft implantation (8). Regrowth ceases as soon as axons contact the CNS milieu again. Various studies have demonstrated that central axons can elongate within autologous PN grafted into the spinal cord and form functional synapses with skeletal muscles, leading to motor and sensory recovery (7, 9-11). Spinal cord neurons, as well as the midbrain and brainstem neurons that originate the rubrospinal, vestibulospinal, and reticulospinal tracts, are endowed with a high capability of axonal regeneration into PN transplants (12-15). Thus, in an attempt to bypass a spinal cord lesion by connecting descending motor fibers with skeletal muscles, muscular nerve branches were inserted into the severed lateral bundle of monkey spinal cord (11). The new connection produced muscle reinnervation and restored motor function. This result raised the possibility that the regrowth of axons descending from central noncholinergic neurons and cut during the grafting procedure could be re...

Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction

“…The results are in agreement with findings of glutamatergic transmission at adult vertebrate NMJs (34) and with the release of glutamate from neonatal mammalian motorneurons (35,36). Glutamatergic-cholinergic cotransmission at the vertebrate NMJ may contribute to apparently normal behavioral and physiological development after chronic blockade of embryonic electrical activity (37,38).…”

Transmitter-receptor mismatch in GABAergic synapses in the absence of activity

“…In this model, NMDA-and AMPAreceptor expression increases when activity is suppressed and decreases when activity is enhanced (3). Interestingly, under experimental glutamatergic innervation, the rat neuromuscular junction switches from cholinergic-to glutamatergic-type synapse (4). A similar activity-dependent control has been shown in the mature hippocampus.…”

New Aspects of Graft-induced Central Plasticity and Neuroregeneration at T 10 in a Rat Model†

“…Oro-pharyngeal intubation for artificial ventilation was performed only when Vecuronium ® was to be administered with respect the type of reinnervation/ neurotransmission in rats (No 19 NaCl, 21 CERE, 28 CERE, 29 Na Cl). Finally after the incision of the epineurium of IOAM nerve stump Fast Blue ® -soakedpats were attached around the nerve fascicles, distal to the coadaptation ( Figure 3) [11,[35][36][37]40,41].…”

“…min, inspired oxygen fraction (FiO 2 )-0.7) for a duration of 15 to 25 min. A warm circulating water blanket was used intra-and postoperatively to prevent hypothermia by maintain the body temperature of the rats within the physiological range [11,[33][34][35][36].…”

“…This goal has been successfully achieved both in macaques and in paraplegic human restorative surgery by Brunelli's concept of autologous nerve grafting procedures with direct neurotization of paralyzed muscles in 2000-2002 [9][10][11]. It is still difficult to understand Brunelli's paradigm (Figure 1), which has demonstrated that the re-establishment of voluntary isolated muscle activation in humans is not replicated elsewhere, despite strenuous effort over the prior decade focused primarily on transdisciplinary treatment [1,2,12].…”