A method for staining freeze-substituted tissue has been developed which requires no further stain after the sections are cut, so the stain extends evenly through the section. Therefore the three-dimensional structure of the cytoskeleton and related fine filaments in synapses can be determined in continuous serial sections. How neurofilaments end in synaptic terminals has been determined; this is important because neurofilament lengths are thought to be regulated by Ca-activated proteases at their terminations. Growing nerve terminals in the brain have been reconstructed from serial sectioned freeze-substituted preparations. These new preparative methods have revealed an internal system of membranes which are thought to be the source of the new membrane added to the surface of the growth cone during its growth. These membranes are highly labile and are destroyed by conventional fixatives. Current evidence indicates that they participate in recycling of membranes needed for extension of the growth cone as well as function as intracellular compartments where the plasmalemmal surface of the growth cone can be modified during neuronal development. New collaborative work on auditory hair cells with video microscopy shows the distribution there of transmitter enzymes as well as shape changes in living hair cells during transduction. Another collaborative study has used immunofreeze-fracture to show that the distribution of acetycholine receptors in lipid vesicles corresponds to particles protruding from their membranes; this is preliminary to a study of acetycholine receptor structure.
