Fixation effects on synaptic vesicle density in neuromuscular junctions of young and old mice

It was previously reported that in soleus neuromuscular junctions of old mice, synaptic vesicle density was decreased while transmitter release was increased (compared to results in young mice). In the present study, two hypotheses that might resolve this disparity were tested. The first was that the density of readily releasable vesicles close to the preterminal membrane, rather than those in the whole terminal, would correlate with the physiological results. This hypothesis was excluded because both vesicle density in the 200 nm region just within the presynaptic terminal membrane, and total vesicle density were similarly reduced in old soleus junctions. The second hypothesis was that more transmitter was released during fixation at old than at young neuromuscular junctions, leading to an age-related depletion of vesicles. This was tested by counting vesicles in muscles fixed after transmission block was attained in Krebs solution lacking calcium, and by direct recording of quantal release during conventional fixation. This second hypothesis was excluded: in neuromuscular junctions exposed to zero-calcium Krebs solution before fixation, the age-related reduction in vesicle density was still present, and intracellular recording revealed only a slight increase in quantal transmitter release during fixation. Therefore, as discussed, other mechanisms must be considered.