Focal adhesions to the synaptic matrix at mouse neuromuscular junctions

Abstract

Studies of identified synapses in living animals have revealed ongoing synaptic remodelling in the mature nervous system. We recently demonstrated the existence of filopodia and lamellipodia (structures associated with nerve outgrowth in developing or tissue culture systems) at the mature mouse neuromuscular junction (NMJ) . Evidence of motor nerve terminal retraction, regeneration and perisynaptic outgrowth indicated that even established synaptic contacts were not permanent. It now appears that variation in the stability of synaptic contacts, not the amount of nerve terminal outgrowth,is the basis for synaptic remodelling and extension. Although it has been documented that regenerating motor nerve terminals adhere to former synaptic matrix,and that the matrix contains information for inducing and aligning presynaptic and postsynaptic specializations,it was not clear whether the synaptic matrix was uniformly adhesive, or formed specialized adhesive foci with the preterminal membrane. To determine if there are specific sites of synaptic adhesion we utilized hypertonic fixatives to induce shrinkage at the NMJ.Male mice were anaesthetized, the soleus muscle was exposed, and the overlying skin was retracted to create a pool into which either 2% glutaraldehyde in HEPES Krebs (normal fixative) or 2% glutaraldehyde in HEPES Krebs with a 2X NaCl concentration (hypertonic fixative) was added continually for 30 minutes. The surface layer of each muscle was then removed and submerged in the same fixative for an additional 1.5 hours. Tissue was washed in buffer (pH 7.2) and immersed in cholinesterase stain for 5 minutes to localize endplate regions . Endplate regions were cut into 1 mm blocks, washed in buffer (pH 7.2) and post-fixed for 1 hour in HEPES Krebs buffered 2% osmium tetroxide (pH 7.2). Following fixation, specimens were dehydrated in a graded ethanol series and embedded in Polybed 812 (Polysciences). Mouse soleus muscle was stained for immunocytochemical localization of actin with a rabbit polyclonal antiserum against chicken gizzard actin (from Dr. James Lessard, University of Cincinnati) as previously described .