Nitric Oxide Mediated Signal Transduction in Networks of Human ...

of the pre-synaptic vesicle associated proteins (synapsin I and synaptotagmin I). The staining of
both proteins appeared as punctate along the neurites and increased significantly within 2-4 weeks
of in vitro culture indicative of pre-synaptic maturation (Figs 1&2, Tegenge et al., 2009). Evidence
for activity-dependent pre-synaptic vesicle release was provided by employing antibody against the
luminal domain of synaptotagmin I. Upon fusion of synaptic vesicles with the plasma membrane,
the luminal domain of synaptotagmin becomes exposed to the neuronal surface, allowing the
binding of the antibody and their subsequent internalization through the endocytosis of synaptic
vesicle (Figure 2a). In human NT2 neurons I demonstrated that luminal synaptotagmin antibody is
incorporated in both depolarization and calcium dependent manner, which suggest that the neurons
display synaptic vesicle recycling. In NT2 neurons cultured only for 1 week we hardly detected
luminal synaptotagmin punctate staining compared to intense staining within 2-4 weeks. Thus,
activity-dependent incorporation of luminal synaptotagmin antibody depends on the length of in
vitro culture of the neurons indicating that human NT2 neurons undergo synaptic maturation
(Tegenge et al., 2009).
Figure 2. Methods to monitor the synaptic vesicle recycling and exocytosis in developing neurons.
(a) Use of antibodies against the luminal domain of the synaptic vesicle protein synaptotagmin (Syt-
14