Faculty Bibliography

Synapsin I, a neuron-specific, synaptic vesicle-associated phosphoprotein, is thought to play an important role in synaptic vesicle function. Recent microinjection studies have shown that synapsin I inhibits neurotransmitter release at the squid giant synapse and that the inhibitory effect is abolished by phosphorylation of the synapsin I molecule (Llinas et al., 1985). We have considered the possibility that synapsin I might modulate release by regulating the ability of synaptic vesicles to move to, or fuse with, the plasma membrane. Since it is not yet possible to examine these mechanisms in the intact nerve terminal, we have used video-enhanced microscopy to study synaptic vesicle mobility in axoplasm extruded from the squid giant axon. We report here that the dephosphorylated form of synapsin I inhibits organelle movement along microtubules within the interior of extruded axoplasm and that phosphorylation of synapsin I on sites 2 and 3 by calcium/calmodulin-dependent protein kinase II removes this inhibitory effect. Phosphorylation of synapsin I on site 1 by the catalytic subunit of cAMP-dependent protein kinase only partially reduces the inhibitory effect. In contrast to the inhibition of movement along microtubules seen within the interior of the axoplasm, movement along isolated microtubules protruding from the edges of the axoplasm is unaffected by dephospho-synapsin I, despite the fact that the synapsin I concentration is higher there. Thus, synapsin I does not appear to inhibit the fast axonal transport mechanism itself. Rather, these results are consistent with the possibility that dephospho-synapsin I acts by a crosslinking mechanism involving some component(s) of the cytoskeleton, such as F-actin, to create a dense network that restricts organelle movement. The relevance of the present observations to regulation of neurotransmitter release is discussed

The oscillation of membrane potential in mammalian central neurons is of interest because it relates to the role of oscillations in brain function. It has been proposed that the entorhinal cortex (EC), particularly the stellate cells of layer II (ECIIscs), plays an important part in the genesis of the theta rhythm. These neurons occupy a key position in the neocortex-hippocampus-neocortex circuit, a crucial crossroad in memory functions. Neuronal oscillations typically rely on the activation of voltage-dependent Ca2+ conductances and the Ca2+ -dependent K+ conductance that usually follows, as seen in other limbic subcortical structures generating theta rhythmicity. Here we report, however, that similar oscillations are generated in ECIIscs by a Na+ conductance. The finding of a subthreshold, voltage-gated, Na+ -dependent rhythmic membrane oscillation in mammalian neurons indicates that rhythmicity in heterogeneous neuronal networks may be supported by different sets of intrinsic ionic mechanisms in each of the neuronal elements involved

The question as to whether synaptic vesicles prepared from vertebrate brain can be transported to the active zones of the squid giant synapse was studied by using a combined optical and electrophysiological approach. In order to visualize the behavior of the vertebrate synaptic vesicles in situ, synaptic vesicles isolated from rat brain were labeled with a fluorescent dye (Texas red) and injected into the presynaptic terminal of the squid giant synapse. The pattern of fluorescence that would result from passive diffusion was determined by coinjection of an unconjugated fluorescent dye (fluorescein). The patterns obtained with fluorescent synaptic vesicles were strikingly different from that obtained by simple diffusion of fluorescein. Although the fluorescein diffused freely in both directions, the vesicles moved preferentially into the terminal--i.e., toward the release sites--at a rate of 0.5 microns/sec. The final distribution of the injected fluorescent synaptic vesicles displayed a discrete localization that suggested a distribution coincident with the active zones of the presynaptic terminal. Like fast axonal transport, but unlike fluorescein movements in the terminal, the vesicle movement was energy dependent, since the addition of 2,4-dinitrophenol blocked the redistribution of vesicles completely. In addition, reduction of extracellular calcium concentration reversibly blocked vesicular movement as well. In conclusion, mammalian synaptic vesicles retain the cytoplasmic surface components necessary for translocation, sorting, and targeting to the proper locations by the native machinery of the squid giant synapse