Ph.D Thesis

Ph.D StudentVardinon-Friedman Hagit
SubjectThe Assembly of Synapses Formed among Neurons in the
Mammalian Central Nervous System
DepartmentDepartment of Medicine
Supervisor PROF. Noam Ziv


The mammalian brain is a complex network of computational units - neurons - interconnected by specialized intercellular communication devices - synapses. Most synapses are formed between axons (major carriers of neuronal output), and dendrites (major targets for neuronal input). Synaptic connectivity requires the formation of unique specializations at the pre and postsynaptic compartments at sites of direct cell-to-cell contact. Synapse formation begins with the formation of a physical contact between potential synaptic partners, continues with the differentiation of the axonal and dendritic compartments into pre- and postsynaptic compartments respectively, and may be considered completed when the nascent junctions acquire a capacity for synaptic transmission. 

My research was aimed at determining the overall time frame of the formation of individual glutamatergic synapses between mammalian CNS neurons, the temporal order of key events during the assembly of individual glutamatergic synapses (timing of Active Zone formation, Post Synaptic Density (PSD) assembly, recruitment of glutamate receptors, accumulation of synaptic vesicles and the development of synaptic functionality), the modes by which pre and postsynaptic assembly is realized, and to examine the importance of activity of NMDA receptors and N-cadherin adhesion molecules during the formation of CNS synapses.

To that end we utilized an experimental model system based on developing primary cultures of rat hippocampal neurons, automated time-lapse, multi-site laser scanning confocal microscopy, vital fluorescent labels, and immunohistochemistry.

Our findings suggest that new glutamatergic synapses can form within ~2 hours or less. We found that new, apparently functional presynaptic sites may form within 30 minutes of axodendritic contact, and that such sites are practically always already associated with two presynaptic structural molecules, bassoon and piccolo. Presynaptic functional boutons seemed to achieve functional status slightly before the postsynaptic reception apparatus, as PSD proteins seemed to show a more gradual recruitment to new axodendritic contacts. Specifically, clustering of ProSAP1/Shank2, ProSAP2/Shank3 and SAP90/PSD-95 seems to take place concomitantly with or after maturation of presynaptic compartment, while another PSD molecule (GKAP) seemed to be recruited later. Further analysis revealed that within less than 90 minutes of their initial appearance, most new presynaptic sites were gradually associated with all postsynaptic molecules examined. NMDA and AMPA receptors were recruited to new synapses with similar kinetics, and NMDA receptor activation did not seem to be required for the recruitment of AMPA receptors to new synaptic junctions. Finally, we did not find evidence that N-cadherin plays a crucial role in synapse formation.

These findings provide important new information on the whole process of synapse formation and on the roles of key synaptic proteins in the assembly process of new glutamatergic synapses in the developing central nervous system.