Mature and Juvenile Neuromuscular Plasticity in Response to Unloading

Abstract

The neuromuscular junction (NMJ) is the synapse that enables the requisite electrical communication between the motor nervous system and the myofibers that respond to such electrical stimulation with movement and force development. Changes in an NMJ's normal activity pattern have been demonstrated to remodel both the synapse and the myofibers that comprise the NMJ. Significant amounts of research have been devoted to the study of aging on the neuromuscular system. Far less, however, has been focused on revealing the effects of reduced activity on the NMJ and myofibers comprising juvenile neuromuscular systems. In the present investigation, the consequences of decreased activity imposed by muscle unloading (UL) via hindlimb suspension for 2 weeks (a period known to induce muscle remodeling) were examined in both young adult, that is, mature (8 mo), and juvenile (3 mo) neuromuscular systems. In total, 4 treatment groups comprised of 10 animals (Juvenile-Control, Juvenile-Unloaded, Mature-Control, and Mature-Unloaded) were studied. Immunofluorescent procedures, coupled with confocal microscopy, were used to quantify remodeling of both the pre- and postsynaptic features of NMJs, as well as assessing the myofiber profiles of the soleus muscles housing the NMJs of interest. Results of ANOVA procedures revealed that there were significant (p < 0.05) main effects for both treatment, whereby UL consistently led to expanded size of the NMJ, and Age where expanded NMJ dimensions were consistently linked with mature compared to juvenile neuromuscular systems. Moreover, only sporadically was interaction between the main effects of Age and Treatment noted. Importantly, one variable that remained impressively resistant to the effects of both Age and Treatment was the critical parameter of pre- to postsynaptic coupling suggesting stability in effective communication at the NMJ throughout the lifespan and despite changes in activity patterns. The data presented here suggest that further inquiry must be performed regarding disuse-related plasticity of the neuromuscular system in adolescent individuals as those individuals regularly suffer injuries resulting in periods of muscle UL.

Keywords: adolescence; aging; atrophy; disuse; synapse.

FIGURE 1

Representative immunofluorescent staining of the NMJ. Presynaptic nerve terminal branches are specifically visualized with the RT‐97 primary antibody conjugated with secondary antibody labeled with Alexa Fluor 488 (green), and postsynaptic ACh receptors are identified with bungarotoxin labeled with Alexa Fluor 555 (red). Scale bar = 20 µm.

FIGURE 2

Representative immunofluorescent staining of the NMJ. In this example, presynaptic ACh‐containing vesicles are displayed with the SV‐2 primary antibody, which is tagged with secondary antibody conjugated to Alexa Fluor 647 (blue), and postsynaptic ACh receptors are identified with Alexa Fluor 555 (red). Scale bar = 10 µm.

FIGURE 3

Representative example of tracings for total area and total perimeter length of NMJ are seen with green line encompassing entire structure. Stained area and stained perimeter length are seen with yellow line collectively encompassing entire stained area and entire stained length. Scale bar = 20 µm.

FIGURE 4

Representative immunofluorescent staining of NMJ pre‐ to postsynaptic coupling. Presynaptic nerve terminal branches are identified with the RT‐97 primary antibody with secondary antibody conjugated with Alexa Fluor 488 (green), and postsynaptic ACh receptors are labeled with bungarotoxin labeled with Alexa Fluor 555 (red). Scale bar = 20 µm.

FIGURE 5

Overlay of presynaptic vesicles with postsynaptic receptors of the NMJ. Coupling is determined as percentage of postsynaptic endplate area also occupied by presynaptic vesicles. Scale bar = 10 µm.

FIGURE 6

Representative immunofluorescent staining of myofibers. Type I fibers are visualized with Alexa Fluor 555 (red), and Type IIA fibers are stained with Alexa Fluor 350 (blue) secondary antibodies. Type IIB fibers are not expressed in the soleus muscle of the mature rat, and only very few Type IIX fibers were present (remaining unstained). Scale bar = 100 µm.