Synaptogenesis refers to all the processes involved in the formation of synapses [1]. This multi-step process is crucial in neurodevelopment in early stages of life and continues to play a vital role in learning, plasticity, and adaptation throughout life. In neurodevelopment, this phase occurs following axonal migration at which point it is believed that differentiation occurs in the presynaptic terminal, the synaptic cleft, and the postsynaptic reception [1]. While the multi-step process is complicated and involves many players, the entire neurodevelopment phase can be classified into three general stages: cell adhesion, pleomorphic clustering, and pre-/post- synaptic differentiation and specialization [1]. The proteins fall under the goals of synaptic assembly and synaptic specificity [2].


1. Overview
1.1 Synaptogenesis in Neural Development
1.2 Synaptic Assembly and Formation
2. Synaptic Assembly
2.1 Cell-Cell Adhesion and Pleomorphic Clustering
3. Synaptic Formation
3.1 Synaptic Differentiation
3.1.1. Pre-synaptic Differentiation
3.1.2. Post-synaptic Differentiation
4. References

1. Overview

1.1 Synaptogenesis in Neural Development

Synaptogenesis has its peak activity at 18 weeks of gestation and continue to maintain high levels of activity into childhood. Although the activity tapers off with age, activity-dependent synaptic plasticity persists throughout life [3

Neural development consists of seven stages [4]:

1. Cell birth (neurogenesis, gliogenesis)
2. Cell migration
3. Cell differentiation
4. Cell maturation (dendrite and axon growth)
5. Synaptogenesis (formation of synapses)
6. Cell death and synaptic pruning
7. Myelogenesis (formation of myelin)

These stages do not occur in isolation or in linear progression. Instead the stages are dependent on different chemical cues to initiate, terminate, and repeat [4]. First, neuro-ontogenic process begins at gestational age around weeks 2-3 [3]. Between cortical patterning and neuronal differentiation, a series of overlapping cellular events occur as specialization and functionality takes place [3]. Neuronal migration and axonal pathfinding peaks between gestational age weeks 12 to 20 until around weeks 26 to 29 [3]. Synaptogenesis begins among neurons that have reached their target areas and initiates with early synaptic connections [3]. The earliest synaptic connections have been observed around gestation age week 5and reaches a peak period at gestational age week 34 where about 40 000 new synapses are formed every second [3]. Synaptic maturation is regulated by many proteins involved in synaptogenesis and is also governed by activity-dependent changes and hormones [3].

1.2 Synaptic Assembly and Formation

Time frames during which synaptogenesis occurs is different between cortical layers however the mechanisms of the process is the same, especially during the period of neural development [5]. Two phases define synaptogenesis: synaptic assembly and synaptic formation [2]. Synaptic assembly includes all the processes that aid in gathering necessary components for synaptic formation [2]. Some proteins expressed in other stages such as during axonal finding have been documented to play additional signaling roles to initiate localization of proteins that function at the synapse [2].
Synaptic formation then refers to the processes and mechanisms that contribute to the differentiation and maturation of the synapse [2]. Specifically, the pre-synaptic terminal and the post-synaptic terminal have different components that aid in their differentiation [2]. Furthermore, separate proteins are recruited to facilitate inhibitory neuronal connections and excitatory neuronal connections which are key in learning and memory [2].

2.Synaptic Assembly
The initial steps in synaptic assembly are to ensure cell contact and the maintenance of the connection. The Eph family has been described in axonal finding to be a family of molecules that aid in growth cone guidance [3]. At the target location, other proteins are recruited for cell-cell adhesion by factors such as ephrin-B [3]. A few of they vital components of cell-cell adhesion are reported here with the understanding that there are many known and unknown others.

Cell-Cell Adhesion components
Multiple components exist in the activity of cell-cell adhesion and while they all function for the same goal, they can sometimes operate independent of each other.

CAM (Cell adhesion molecules)
Cadherins are family of CAMs that have an important role in the assembly of synapses [1]. A subclass of cadherins is classical cadherins which includes the neural cadherin, N-cadherin [1]. It appears at the synaptic site spontaneously as the mechanisms of cadherin recruitment are not entirely clear. Furthermore it is understood that the adhesive properties of cadherins aid in the localization to the area of cell-cell adhesion and to direct synaptic morphology however, these mechanisms remain to be clarified.

Integrins make up another family of adhesion molecules consisting of surface glycoproteins and consists of a range of extracellular matrix molecules for ligands [6]. Integrins have a role in both postnatal brain development and the adult brain function [6]. Their role in postnatal development consists of interactions with laminin and signaling to facilitate PSD [6]. The absence of integrin, specifically beta-integrin, is marked with decreased cell-cell adhesion at premature synaptic sites and increased migration of glial cells from their marked targets [6].

The ACh Receptor is a postsynaptic receptor that clusters in high density concentration areas. The clustering of AChR has been found to form prior to cell-cell contact and prior to any associations with the synaptic partner [1]. The function of the observed clustering has been observed to be part of synaptic assembly in that the AChR aggregation aids in the stability of filopodia until the physical adhesion process [1]. Furthermore, AChR has been observed to be associated with scaffold protein complexes containing PSD-95, GKAP, Shank, and neuroligin 1 (NLG-1) at the predefined synaptic location [1]. At contact, immediate recruitment of synaptophysin and other synaptic formation protein-containing vesicles ensues [1].

Abi-1 has two distinct applications in the process of synaptogenesis. During early neuronal development, Abi-1 is localized in the neurites and growth cones to aid the enrichment of dendritic spines and post synaptic densities [7]. Interacting with ProSAP2/Shank3 (post-synaptic density proteins), Abi-1 can be detected in the dendrites at PSD areas of excitatory synapses [7]. In hippocampal neurons, the presence of Abi-1 facilitates the production of actin cytoskeleton in neurites and in growth cones with high actin turnover [7]. It has been shown that the activity of Abi-1 in hippocampal neurons stabilizes actin filaments and cell adhesion to facilitate further synaptic formation and maturation [7]. The absence of Abi-1 resulted in outgrowth of immature dendrites and filopodia [7]. Abi-1 up to this stage is considered one of the trigger proteins to progress further into synaptic formation, maturation, and differentiation.
In addition, dendritic structures are dependent upon physiological levels of Abi-1 - therefore indicating that the expression of Abi-1 is highly regulated since an over expression or under expression of the protein disrupts the formation of dendrites [7]. The trimeric complex of Abi-1, Eps8, Sos-1 translocates to the nuclei when the NMDA protein is applied [7].

Neurexin-Neuroligin Complex
The most important cell-cell adhesion complex is that formed between the presynaptic protein beta-neurexin (NRX) and neuroligin-1(NGL1) [8]. As a key trans-synaptic adhesion complex in the initial synaptic assembly phase, the complex operates to stabilize the trans-synaptic connection on the extracellular surface [8]. It further operates as a membrane-based stabilizer for the purpose of providing tension and traction at the synapse. The complex functions to maintain adhesion and stabilize filopodia so that there is a stable increase in synaptic density and synaptic vesicle clustering [8]. Mutations in either of the binding partners have been recently identified to contribute to autism spectrum disorder [8]. On the intercellular surface, the binding of each molecule induces pre and post-synaptic differentiation through the signaling and recruitment of synaptic proteins [8]. Throughout synaptogenesis, the complex continues to maintain cell-cell adhesion on top of gaining other functions in maturation, recruitment, and specialization of the synapse.
The role of NLG1 is further emphasized by its importance in recruiting the right amount of post-synaptic protein to the synapse [9]. Found on the surface of young neurons, NLG1 accumulates in clusters and is also mobile in these clusters [9]. The accumulation then to the synaptic site occurs by the cue of axonal contact and can be observed to have formed larger clusters at an average of 75s [9]. Further recruitment - at a much slower rate of 8.1 minutes - is facilitated by the interactions with beta-NRX. This crucial interaction has been suggested to be the initial step in synaptogenesis [9].

3.1 Synaptic Differentiation
Both pre- and post- synaptic terminals recruit proteins, vesicles, and necessary molecules for differentiation and specialization through the use of already localized proteins and receptor domains however the mechanisms as to how this is achieved is drastically different. Furthermore there are few, if any, proteins active in synaptic differentiation that are present in both terminals.

List of (some of the) known proteins involved in pre and post synaptic organization [1]

3.1.1. Pre-synaptic Differentiation

The presynaptic terminal is strongly indicated by the presence of presynaptic active zones (AZs) [10]. This area makes up the platform for the rapid fusion of neurotransmitter-filled synaptic vesicles after activation through calcium influx [10]. As such this zone is electron dense and strongly associated with the cell membrane [10]. In mediating neurotransmitter release, the vesicles carrying those neurotransmitters are then recycled in the areas adjacent to the AZs [11].

Pre-synaptic Protein clusters [11]

It is noted that the process of synaptogenesis more often than not invovles multi-step processes with protein complexes. The set-up of the presynaptic terminal can be grouped into three main complexes at the AZ [11]. One of the complexes includes SNARE in conjunction with VAMP and SNAP25 [11]. The main function of this complex is to assist vesicles in docking and fusing to the membrane [11]. A second major complex observed was that of Munc18, Munc13, and synaptotagmin. Although not always seen to interact with each other or clustered in a complex, this group of proteins collaborates with SNARF to regulate excytosis of vesicle and other content [11]. Finally the third protein complex consists of piccolo, bassoon, RIMS, Liprin, CAZ, and Mints [11]. This set of molecules, in addition to aiding exocytosis and endocytosis, also tethers and organizes the vesicles for the aforementioned purpose [11].

In presynaptic differentiation, there is great reliance upon receptor localization and molecule recruitment. This rests further on synaptic vesicle maturation through the usage of synaptic vesicle precursors [12]. The more than fifty synaptic vesicle proteins are derived from two precursor vesicles that later mature into functional synaptic vesicles after many rounds of recycling and maturation. These are formed at the level of the Golgi and can be detected before even the vesicle clusters [12].
Further details of presynaptic differentiation, specialization, and mechanisms involved can be found at presynaptic plasticity.

3.1.2. Post-synaptic Differentiation

Just as the presynaptic terminal is markedly characterized by the active zone, the post-synaptic terminal area is governed by the post-synaptic density (PSD) [12]. The PSD is characterized by neurotransmitter receptor accumulation with emphasis on NMDA and AMPA receptors [13]. The PSD aids in further establishing stable and dynamic regulatory control over the receptors and proteins that are localized and colocalized to the area [14].

Other proteins seen before such as Abi-1 becomes translocated from the membrane to the nuclei with the activity of NMDA at the post-synaptic terminal [7]. Its new function is to bind with transcriptional factors Myc/Max in the E-box to regulate EGF receptor and GABA-A receptor expressions [7, 14]. The regulation represses the extension of immature synaptic outgrowth and tailors the post-synaptic formation towards specialization and maturation [7].
NLG1, located in the post-synaptic terminal has a intercellular post-synaptic density zone (PDZ) domain that interacts with PSD-95, a well-documented and researched post-synaptic differentiation and functional scaffold protein which helps to facilitate both clustering and maturation [14]. Both PSD-95 and NMDAR are localized to the PSD by the NLG1 activity however they are done so via separate pathways [13]. Finally the action of NLG1 is accelerated by thrombospondin 1 (TSP1) [15]. This is an important molecule in terms of synaptic regulation because it is only present in earlier stages – in younger neurons to facilitate outgrowth of premature synapses (filopodia) [15]. Prolonged expression of TSP1 has been considered a cause of concern in mental retardation and other neurological disorders [15].
Immature synapses exhibit highly sensitive reactions to levels of Zn2+ ions [15]. This sensitivity is strongly linked with ProSAP1/Shank2, ProSAP2/Shank3, but not Shank1 [16]. This interaction with Zn2+ ions at the post-synaptic terminal further demonstrates the control of immature synapses through environmental sensitivities that give way to PSD proteins [16]. The desensitization of the post-synaptic terminal to Zn2+ ions comes concurrently with increased post-synaptic activity of the ProSAP/Shank family thus indicating their key regulatory role in the formation, stabilization, and maturation of excitatory synapses [16].

The mechanisms and further specialization of the post-synaptic terminal is elaborated with the inspection of the post-synaptic plasticity.

6. References

1. Garner, C.C., Zhai, R.C. Gundelfinger, E.D., Ziv, N.E. (2002) Molecular Mechanisms of CNS Synaptogenesis. Cell Press. 25-5. 243-250
2. Colon-Ramos, D.A. (2009) Synapse Formation in Developing Neural Circuits. Current topics in Developmental Biology 87. 53-80
3. Tau, G.Z., Peterson, B.S. (2010) Normal Development of Brain Circuits Neuropsychopharmacology 35. 147-168
4. Kolb, B., Gibb, R. (2011) Brain Plasticity and Behaviour in the Developing Brain. J Can Acad Child Adolesc Psychiatry.20 (4). 265-276
5. Ziv, N.E., Garner, C.C. Presynaptic Development and Active Zones. (2009) Elsevier 957 - 967
6. Nikonenko, I., Toni, N., Moosemayer, M., Shigeri, Y., Muller, D., Jones, L.S. (2002) Integrins are involved in synaptogenesis, cell spreading, and adhesion in the postnatal brain. Developmental Brain Research. 140: 185-194
7. Proepper, C., Johannsen, S., Liebau, S., Dahl, J., Vaida, B., Bockmann, J., Kreutz, M.R., Gundelfinger, E.D., Boeckers, T.M. (2007) Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation. The EMBO Jounral 26. 1397-1409
8. Chen, S.X., Tari, P.K., She, K., Haas, K. (2010) Neurexin-Neuroligin Cell Adhesion Complexes contribute to synaptotropic dendritogenesis via growth stabilization mechanisms in vivo. Neuron 67: 967-983
9. Barrow, S.L., Constable, J.R.L., Clark, E., El-Sabeawy, F., McAllister, A.K., Washbourne, P. Neuroligin 1: a cell adhesion molecule that recruits PSD-95 and NMDA receptors by distinct mechanisms during synaptogenesis. Neurodevelopment 4: 16 - 44
10.Owald, D., Sigrist, S.J., (2009) Assembling the presynaptic active zone. Current Opinion in neurobiology 19:311-318
11. Zhen, M., Jin, Y. (2004) Presynaptic terminal differentiation: transport and assembly. Current Opinion in Neurobiology 14:280-287
12. Harrill, J.A., Robinette, B.L., Mundy, W.R. (2010) Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro. Toxicology in Vitro 25: 386-387
13. Zheng, S., Gray, E.E., Chawla, G., Porse, B.T., O'Dell, T.J., Black, D.L. (2012) PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2 Nature Neuroscience 15(3):381-290
14. Patrizi, A., Scelfo, B., Viltono, L., Briator, F., Fukaya, M., Watanabe, M., Strata, P., Varoqueaux, F., Brose, N., Frischy, JM, Sassoe-Pognetto, M.(2008) Synapse Formation and Clustering of Neuroligin-2 in the Absence of GABA-A Receptors Proceedings of the National Academy of Sciences of the United States of America , 105 (35) 13151-13156
15.Xu, J., Xiao, N., Xia, J. (2010) Thrombospodin 1 accelerates synaptogenesis in hippocampal neurons through neuroligin 1. Nature Neuroscience 13(1):22 – 25
16.Grabruker, A.M., Knight, M.J., Proepper, C., Bockmann, J., Joubert, M., Roawn, M., Nienhaus, G.U., Garner, C.C., Bowie, J.U., Kruetz, M.R., Gundelfinger, E.D., Boeckers, T.M. (2011) Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation. The EMBO Jounral 30: 569-581

The initial steps in synaptic assembly are to ensure cell contact and the maintenance of the connection. The Eph family has been described in axonal finding to be a family of molecules that aid in growth cone guidance [3]. At the target location, other proteins are recruited for cell-cell adhesion by factors such as ephrin-B [3]. A few of they vital components of cell-cell adhesion are reported here with the understanding that there are many known and unknown others.