Role of NCAM During Initial Stages of Mammalian Synaptogenesis

To evaluate the roles of NCAM in formation and stabilization of initial contacts, we performed time-lapse recordings in dissociated hippocampal cultures48. NCAM was visualized by indirect immunofluorescence, and TGN-derived organelles were loaded with a fluorescent dye, FM4-64. Within minutes of establishment of physical contacts between the growth cone and target neurite, NCAM immunoreactive clusters and associated organelles that had moved along the target neurite began to accumulate at sites of contact. The clusters and associated intracellular aggregates often passed several times through the actual site of contact until one or several clusters and associated organelles were "trapped" at the contact site and remained there until the end of the recordings (10-100 min) (Figure 6.4). Only occasionally did the growth cone contact the target neurite at the site of an NCAM immunoreactive cluster. In this case, the NCAM immunoreactive clusters and associated intracellular aggregates remained at the site of contact from the moment of its formation. These data suggest that the location of initial contact is not predetermined by NCAM expression, but NCAM may be quickly recruited - together with intracellular organelles - to initial contacts in a quantal manner.

Figure 6.4. Synaptic Trapping of TGN-derived Intracellular Organelles by NCAM. Location of pre- and postsynaptic structures, NCAM clusters, and associated organelles are shown before (A) and a few minutes after (B) initial contact formation.

To investigate whether pre- or postsynaptic NCAM plays a role in the stabilization of organelles at sites of contact, we used the heterogenotypic co-culture model described above35. In co-cultures of wild-type and NCAM-deficient neurons maintained for 4 days in vitro, y-adaptin immunoreactive TGN organelles were also significantly more often associated with contact sites between heterogenotypic axons and dendrites when compared to NCAM negative contacts, showing that heterophilic interactions of NCAM become apparent in a choice situation, i.e., in the presence of NCAM-expressing and NCAM-deficient neurons. TGN organelles were even more often associated with sites of contacts formed by NCAM positive axons and dendrites, implicating a possible contribution of homophilic transinteractions. Time-lapse recordings showed that contacts between NCAM-deficient neurons were more often disrupted due to retraction of neurites when compared to wild-type neurons. Moreover, organelles moved away from contact points approximately four times more often in NCAM-deficient neurons compared to wild-type neurons. Thus, these data demonstrate that NCAM is important for stabilization of initial contacts and recruitment of intracellular organelles to these contact sites.

Figure 6.4. Synaptic Trapping of TGN-derived Intracellular Organelles by NCAM. Location of pre- and postsynaptic structures, NCAM clusters, and associated organelles are shown before (A) and a few minutes after (B) initial contact formation.

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