R430 – Glycogen Depletion for Study of Neuromuscular Connectivity

Problem A useful technique for characterizing connectivity between nerve and muscle involves prolonged electrical stimulation of nerve fibers to deplete glycogen in their target muscle fibers. Depleted muscle fibers appear blank when stained by the PAS technique. Unfortunately, results are inconsistent over a wide range of stimulus paradigms. The aim of this study was to identify other factors that may impact glycogen depletion. Methods Glycogen depletion was examined in the rat medial gastrocnemius muscle because of the presence of aerobic fiber types I and IIA, which are resistant to depletion. Tension and EMG were monitored during maximal stimulation of transected sciatic nerve with a 333 msec pulse train delivered at 40 Hz every second over a period of 1 hour. Once an effective paradigm was identified, depletion of an entirely aerobic muscle (i.e., soleus) was evaluated. Results Animals maintained in a light plane of anesthesia with a steady rate of pentobarbital IP infusion showed an average depletion of only 72%. Animals administered a progressively increasing level of anesthesia with continued stimulation until complete loss of muscular response showed a significantly greater depletion of 96%. However, when this paradigm was applied to the soleus, only 37% of the muscle depleted. Further investigation determined that co-contraction of gastrocnemius with soleus resulted in unloading of the slower soleus. Disruption of the gastrocnemius insertion on the Achilles tendon shifted the load to the soleus and increased soleus depletion to 93%. Conclusion Muscle fibers of any motor unit type can be identified with 93–96% accuracy when nerve stimulation is applied to an isometrically loaded muscle that is in a state of progressive barbiturate anesthesia. Significance Glycogen depletion provides a method for identifying the number, types, and distribution of muscle fibers in a muscle that have reinnervated, important in the assessment of nerve regeneration. Support NIH grants R01 DC001149 and R01 DC008429.

Semaphorin-1a Acts in Concert With the Cell Adhesion Molecules Fasciclin II and Connectin to Regulate Axon Fasciculation in Drosophila

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. Here, by manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, we provide further evidence that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons.

Neuromuscular target recognition by a homophilic interaction of connectin cell adhesion molecules in Drosophila

Drosophila Connectin (CON) is a cell surface protein of the leucine-rich repeat family. During the formation of neuromuscular connectivity, CON is expressed on the surface of a subset of embryonic muscles and on the growth cones and axons of the motoneurons that innervate these muscles, including primarily SNa motoneurons and their synaptic targets (lateral muscles). In vitro, CON can mediate homophilic cell adhesion. In this study, we generated transgenic lines that ectopically expressed CON on all muscles. In the transformant embryos and larvae, SNa motoneurons often inappropriately innervated a neighboring non-target muscle (muscle 12) that ectopically expressed CON. Furthermore, the ectopic synapse formation was dependent on the endogenous CON expression on the SNa motoneurons. These results show that CON can function as an attractive and homophilic target recognition molecule in vivo.