Synaptogenesis is the formation of synapses between neurons in the nervous system. Although it occurs throughout a healthy person's lifespan, an explosion of synapse formation occurs during early brain development, known as exuberant synaptogenesis. Synaptogenesis is particularly important during an individual's critical period, during which there is a certain degree of synaptic pruning due to competition for neural growth factors by neurons and synapses. Processes that are not used, or inhibited during their critical period will fail to develop normally later on in life.
Exuberant synaptogenesis
Brain growth and development begins during gestation and into the postnatal period. Brain development can be divided into stages including: neurogenesis, differentiation, proliferation, migration, synaptogenesis, gliogenesis and myelination, and apoptosis and synaptic pruning. Synaptogenesis occurs in the third trimester during gestation as well as the first two years postnatal. These growth cones find signal molecules which act as guidance cues and form synapses. Connections formed between neurites may be random or selective.
Exuberant synaptogenesis is characterized by a few characteristics. First, it involves the formation of long axonal projections, and an overproduction of small axonal branches, synapses, and dendritic branches and/or spines. Throughout this process, many of these structures may be maintained or eventually eliminated. Elimination may occur by neuronal death or selective deletion.
Developmental exuberance may occur macro- or microscopically. Macroscopic exuberance occurs when transient projections are formed between macroscopic regions in the brain. In comparison, microscopic exuberance occurs when transient structures involved in communication between neurons forms.
Other studies found that this transcription factor was involved in synaptic strength. In this study, it was found that the unc-4 pathway negatively regulates ceh-12, a gene involved in regulating synaptic choice.
Growth cones and guidance cues
thumb|Image of axonal growth cones
Guidance cues are essential for nervous system development as well as synaptic maintenance and remodeling. Guidance cues--attractive or repulsive--are sensed by growth cones. Expression of guidance cue genes is mediated at the transcriptional, post-transcriptional, translational, and post-translational levels.
Most guidance cues converge onto various families of small GTPases which go back and forth from active to inactive forms. There are a multitude of signaling pathways involved in this process but the key ones involve netrins (NTNs) and fibronectin leucine-rich repeat transmembrane proteins (FLRTs), the Slit family, semamorphins (SEMA), ephrin, non-canonical genes (morphogens, chemokines, growth factors), and RTN4 receptors.
Elimination Mechanisms of Transient Projections
In exuberant synaptogenesis, many of the projections formed are eliminated either by neuronal death or selective deletion.
By using retrograde tracing to label transient projections, researchers were able to detect the mechanism of selection axonal deletion. Most of the evidence is provided from studying axonal elimination in the visual cortex, so more research is necessary. However, current research proposes that this elimination mechanism involves retraction of branches over short distances in addition to degeneration of long branches.
The main question that researchers are asking is: what triggers axonal elimination of exuberant synapses? In one study, researchers determined that mice mutant for semaphorin, a molecule that is chemorepulsive to growth cones, had defective pruning in hippocampal mossy fibers. Other chemorepulsive molecules include Slits and ephrins.
Synaptic adhesion molecules (SAMs)
Synaptic adhesion molecules (SAMs) have been presented by researchers as potentially key molecules involved in the organization of synaptic junctions. SAMs are involved in pre- to postsynaptic signaling and the reverse direction.
Distribution
SAMs often form heterophilic complexes that differ based on location. For example, presynaptic SAMs are present on excitatory and inhibitory synapses. In comparison, post synaptic SAMs are very diverse and are specific for excitatory or inhibitory synapses. Neuroligins bind to neurexins. Neuroligin 1 is involved in excitatory specializations formation, but it depends on the results of alternative splicing. Neuroligin 2 is localized to inhibitory synapses. Neuroligin 3 is likely involved in excitatory synaptogenesis, but more research needs to be conducted on this. However, one study found that knockdown of all neuroligins leads to a decrease in frequency of inhibitory but not excitatory miniature synaptic currents.
Another important role of neuroligins and neurexins is the determination of where a synapse forms. For example, co-clustering of neuroligin 1 to PSD-95 acts as a hotspot for presynaptic machinery.
EphBs and Ephrin-Bs
Ephs can be divided into A and B subclasses based on affinity for ephrin-A or ephrin-B ligands. Studies reveal that mainly EphB-ephrin-B interactions are involved in synaptogenesis.
The binding of EphB to Ephrin-B leads to bidirectional signaling and contact-mediated transcellular signaling. During development, this interaction is primarily involved in axon guidance and boundary formation. However, these signaling molecules have also been shown to modify postsynaptic organization.
EphBs are particularly involved in excitatory synaptogenesis. When activated by soluble ephrin-B-Fc fusion protein, EphB induces clustering of NMDARs and AMPARs, an increase in the number of presynaptic terminals, and the formation of dendritic spines. Lastly, binding of Ephrin-B to EphB2 leads to interactions between the extracellular domains of the NMDAR and EphB2.
Immunoglobulins
A key characteristic of Ig molecules is the diverse number of globular extracellular cysteine-looped domains. A number of members of the Ig superfamily have been identified as essential molecules for the organization of pre and post synaptic domains. These include synaptic cell adhesion molecules (SynCAM), synaptic adhesion-like molecules (SALMs), netrin G2 ligand (NGL2), neural cell adhesion molecule (NCAM), etc.
{| class="wikitable"
|Immunoglobulin (Ig) superfamily type
|Function
|-
|Synaptic cell adhesion molecules (SynCAM)
|Regulation of the number of presynaptic specializations, and mediation of cell adhesion independently of calcium.
|-
|Synaptic adhesion-like molecules (SALMs)
|Plays a role in synapse maturation, neurite outgrowth during development, AMPAR clustering, PSD-95-containing synaptic site formation, and the regulation of the formation of excitatory synaptic sites.
|-
|Netrin G2 ligand (NGL2)
|Promotes dendritic spine formation, clustering of PSD-95 and NMDARs, triggering of presynaptic differentiation, formation of excitatory synapses.
|-
|Neural cell adhesion molecule (NCAM)
|Not necessary for synaptogenesis, but hypothesized to play a role in axon guidance.
|}
Cadherins
Neuronal (N)-cadherins are found in pre and postsynaptic terminals. Prior to differentiation, N-cadherins increase in quantity at axon-dendrite contact sites and eventually restrict their presence to sites around the active zone in mature neurons. N-cadherin is also involved in regulating AMPAR trafficking. Besides this, N-cadherin also plays a role in the maturation and stabilization of synaptic specializations. Lastly, N-cadherins help to control dendritic spine morphology and motility.
The mechanisms by which partner choice is determined is also not clear. However, three hypotheses have been proposed to help explain how synapse specificity is determined:
- Partner is choice is determined and then synapse formation occurs, implying they are separate processes mechanistically
- Partner choice and synapse formation are the same process and both are determined by SAMs
- Synapse formation occurs and then a selective elimination process.
However, studies observing a heterologous synapse formation assay and the involvement of SAM in non neuronal cells indicates that hypothesis 1 and 2 are most likely.
Currently, the only SAMs known to be involved in establishing proteins are: postsynaptic adhesion-GPCRs called latrophilins and brain angiogenesis inhibitors. Similarly, teneurins have been presented as mediators in synapse formation.
Properties of Synapses
The properties of synapses is likely shaped by bidirectional signaling between pre- and postsynaptic specialization and are mediated partly by SAMS. This is demonstrated by studies of neurexins, the most common type of SAMs.
Recent studies demonstrate that neurexins are necessary for organizing functional synapses and perform important functions depending on the type of neuron. This is generated by different neurexin isoforms. One example is the difference in function between presynaptic neurexin-1 containing an insert in SS4 (Nrxn1−SS4+) and neurexin-1 lacking an insert in SS4 (Nrxn1−SS4+) generated by alternative splicing. Nrxn1−SS4+ is involved in the trans-synaptic increase in postsynaptic NMDAR levels.
Formation of the neuromuscular junction
The neuromuscular junction (NMJ) is the most well-characterized synapse in that it provides a simple and accessible structure that allows for easy manipulation and observation. Therefore, the synapse is well-researched due to its size and accessibility in the nervous system.
Function
thumb|Image of a neuromuscular junction
The synapse itself is composed of three cells: the motor neuron, the myofiber, and the Schwann cell. In a normally functioning synapse, a signal will cause the motor neuron to depolarize, by releasing the neurotransmitter acetylcholine (ACh). Acetylcholine travels across the synaptic cleft where it reaches acetylcholine receptors (AChR) on the plasma membrane of the myofiber, the sarcolemma. As the AChRs open ion channels, the membrane depolarizes, causing muscle contraction. The entire synapse is covered in a myelin sheath provided by the Schwann cell to insulate and encapsulate the junction. Another important part of the neuromuscular system and central nervous system are the astrocytes. While originally they were thought to have only functioned as support for the neurons, they play an important role in functional plasticity of synapses.
Origin and movement of cells
During development, each of the three germ layer cell types arises from different regions of the growing embryo. The individual myoblasts originate in the mesoderm and fuse to form a multi-nucleated myotube. During or shortly after myotube formation, motoneurons from the neural tube form preliminary contacts with the myotube. The Schwann cells arise from the neural crest and are led by the axons to their destination. Upon reaching it, they form a loose, unmyelinated covering over the innervating axons. The movement of the axons (and subsequently the Schwann cells) is guided by the growth cone, a filamentous projection of the axon that actively searches for neurotrophins released by the myotube. After about a week, a fully functional synapse is formed following several types of differentiation in both the post-synaptic muscle cell and the pre-synaptic motor neuron. This pioneer axon is of crucial importance because the new axons that follow have a high propensity for forming contacts with well-established synapses.
Clustering
AChR experiences multimerization within the post-synaptic membrane largely due to the signaling molecule Agrin. The axon of the motor neuron releases agrin, a proteoglycan that initiates a cascade that eventually leads to AChR association. Agrin binds to a muscle-specific kinase (MuSK) receptor in the post-synaptic membrane, and this in turn leads to downstream activation of the cytoplasmic protein Rapsyn. Rapsyn contains domains that allow for AChR association and multimerization, and it is directly responsible for AChR clustering in the post-synaptic membrane: rapsyn-deficient mutant mice fail to form AChR clusters.
Extrasynaptic repression
Repression of the AChR gene in the non-synaptic nuclei is an activity-dependent process involving the electrical signal generated by the newly formed synapse. Reduced concentration of AChR in the extrasynaptic membrane in addition to increased concentration in the post-synaptic membrane helps ensure the fidelity of signals sent by the axon by localizing AChR to the synapse. Because the synapse begins receiving inputs almost immediately after the motoneuron comes into contact with the myotube, the axon quickly generates an action potential and releases ACh. The depolarization caused by AChR induces muscle contraction and simultaneously initiates repression of AChR gene transcription across the entire muscle membrane. Note that this affects gene transcription at a distance: the receptors that are embedded within the post-synaptic membrane are not susceptible to repression. The NMDA receptor function is associated with the estrogen receptor in hippocampal neurons. Experiments conducted with estradiol show that exposure to the estrogen significantly increases synaptic density and protein concentration.
Synaptic signaling during synaptogenesis is not only activity-dependent, but is also dependent on the environment in which the neurons are located. For instance, brain-derived neurotrophic factor (BDNF) is produced by the brain and regulates several functions within the developing synapse, including enhancement of transmitter release, increased concentration of vesicles, and cholesterol biosynthesis. Cholesterol is essential to synaptogenesis because the lipid rafts that it forms provide a scaffold upon which numerous signaling interactions can occur. BDNF-null mutants show significant defects in neuronal growth and synapse formation. Aside from neurotrophins, cell-adhesion molecules are also essential to synaptogenesis. Often the binding of pre-synaptic cell-adhesion molecules with their post-synaptic partners triggers specializations that facilitate synaptogenesis. Indeed, a defect in genes encoding neuroligin, a cell-adhesion molecule found in the post-synaptic membrane, has been linked to cases of autism and intellectual disability. Finally, many of these signaling processes can be regulated by matrix metalloproteinases (MMPs) as the targets of many MMPs are these specific cell-adhesion molecules. Dendritic spines exhibit three main morphologies: filopodia, thin spines, and mushroom spines. The filopodia play a role in synaptogenesis through initiation of contact with axons of other neurons. Filopodia of new neurons tend to associate with multiply synapsed axons, while the filopodia of mature neurons tend to sites devoid of other partners. The dynamism of spines allows for the conversion of filopodia into the mushroom spines that are the primary sites of glutamate receptors and synaptic transmission.
Contributions of the Wnt protein family
The (Wnt) family, includes several embryonic morphogens that contribute to early pattern formation in the developing embryo. Recently data have emerged showing that the Wnt protein family has roles in the later development of synapse formation and plasticity. Wnt contribution to synaptogenesis has been verified in both the central nervous system and the neuromuscular junction.
Central nervous system
Wnt family members contribute to synapse formation in the cerebellum by inducing presynaptic and postsynaptic terminal formation. This brain region contains three main neuronal cell types- Purkinje cells, granule cells and mossy fiber cells. Wnt-3 expression contributes to Purkinje cell neurite outgrowth and synapse formation. Granule cells express Wnt-7a to promote axon spreading and branching in their synaptic partner, mossy fiber cells. Blocking Wnt expression in the hippocampus mitigates these activity dependent effects by reducing dendritic arborization and subsequently, synaptic complexity.
Adult synaptogenesis in the dentate gyrus
Granule cells formed during the adult period In the dentate gyrus are excitatory neurons that receive glutamate from projection neurons in the entorhinal cortex and mossy cells in the hippocampus, as well as GABA from local interneurons. These neurons have projections to the CA3 region of the hippocampus. A week after these cells are generated, they receive GABA input which is initially depolarizing until two to four weeks when it becomes hyperpolarizing. This is due to inward chloride transporter NKCC1. After the second week, the dendrites of these cells form spines and receive glutamatergic input. By the second month, the electrophysiological and morphological properties of these adult cells is similar to perinatal granule cells.
Research finds that the maturation of adult-born DG granule cells is highly dependent on changes in neuronal activity and most of the new synapses formed by new DG granule cells is the result of seizures. Animal studies reveal that seizures cause increased number of mushroom spines and spiny, branched basal dendrites. Seizures may also cause increased excitability because it causes these cells to fire in synchrony with CA3 pyramidal neurons.
Adult synaptogenesis in the olfactory bulb
Periglomerular neurons
PGNs are classified as GABAergic or dopaminergic modulating interneurons. These receive input from olfactory sensory neurons which project to the dendrites of the primary neurons of the OB. These neurons surround glomeruli that contain olfactory sensory axons that connect to the primary neurons of the OB.
Unfortunately, not much is known about the development of these neurons in the adult brain. However, it was revealed by two-photon imaging that as these neurons mature, the dendritic spines become more stable. Additionally, studies reveal that at the postsynaptic sites of PGNs there are functional changes between sensory neurons and PGNs. For example, at olfactory nerve (ON) synapses there is an increase in the AMPA:NMDA ratio as the brain matures. It is yet to be understood whether this is an intrinsic property of PGNs or if this is due to the continuous turnover of olfactory sensory axons.
Granule neurons
Granule neurons of the OB are axonless GABAergic interneurons which connect to the primary neurons of the OB.