RESUMO
BACKGROUND: In 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. RESULTS: Our 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. CONCLUSIONS: Together, 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.
Assuntos
Regeneração Nervosa , Traumatismos dos Nervos Periféricos , Animais , Camundongos , Regeneração Nervosa/fisiologia , Junção Neuromuscular/fisiologia , Células de Schwann/fisiologia , Sinapses/fisiologiaRESUMO
The neuromuscular junction (NMJ) is the peripheral synapse that controls the coordinated movement of many organisms. The NMJ is also an archetypical model to study synaptic morphology and function. As the NMJ is the primary target of neuromuscular diseases and traumatic injuries, the establishment of suitable models to study the contribution of specific postsynaptic muscle-derived proteins on NMJ maintenance and regeneration is a permanent need. Considering the unique experimental advantages of the levator auris longus (LAL) muscle, here we present a method allowing for efficient electroporation-mediated gene transfer and subsequent detailed studies of the morphology and function of the NMJ and muscle fibers. Also, we have standardized efficient facial nerve injury protocols to analyze LAL muscle NMJ degeneration and regeneration. Our results show that the expression of a control fluorescent protein does not alter either the muscle structural organization, the apposition of the pre- and post-synaptic domains, or the functional neurotransmission parameters of the LAL muscle NMJs; in turn, the overexpression of MuSK, a major regulator of postsynaptic assembly, induces the formation of ectopic acetylcholine receptor clusters. Our NMJ denervation experiments showed complete reinnervation of LAL muscle NMJs four weeks after facial nerve injury. Together, these experimental strategies in the LAL muscle constitute effective methods to combine protein expression with accurate analyses at the levels of structure, function, and regeneration of the NMJ.
RESUMO
Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine.