Synapse Formation (Synaptogenesis) e.g., Neuromuscular Junction

Thompson, R. A. , & Nelson, C. A. (2001) Thompson, R. A., & Nelson, C. A. (2001). Developmental science and the media: Early brain development. American Psychologist, 56(1), 5-15.

A dynamic process. Occurs throughout life. Dependent on learning, experiences, environment, lifestyle, health. The basis for storing information and modulating behavior. Much of what we know about the structure/function of synapses comes from study of the relatively simple synapses outside the CNS: the neuromuscular junction (synapse for Ach transmission).

The axonal ending and target cell membrane initially lock the specializations that make up a synapse. Only upon contact  a new series of instructions occur. E.g., timing: synapse formation begins very early (i.e., 1st few days of a tadpole’s life), but is then protracted (much fine-tuning occurs).

At first, the postsynaptic muscle membrane is nearly equally responsive throughout the entire surface (scattered cluster of Ach receptors). As the neuron approaches the 1st ‘specialization’ there is more precise clustering of these receptors just under the presynaptic boutons.

Skeletal muscle

Neuromuscular junction

Neuromuscular junction synapse: An electronmicroscopic view Pre- and postsynaptic membranes are highly specialized. The nerve terminal is capped by a Schwann cell and is situated in a shallow depression of the muscle cell membrane (postjunctional fold). ACh vesicles are concentrated at the presynaptic site. Rapsyn, neuregulin receptors and muscle specific kinase are concentrated at the postsynaptic site.

Neurotransmitter is released spontaneously from growth cones (next slide) A. The outside-out patch: membrane contained AchRs that were facing outward. The further away the “sniffer” is, the less current passed through the AchRs. When the pipet was brought closer, the AchR-evoked current was larger.

Contact with target increases free Ca2+ in the growth cone (next slide) The muscle-evoked rise in Ca2+ indicates that Ca2+ channels must be involved (Ca2+-free medium).

Characteristics of the Synapse to Develop Postjunctional folds. Active zones in the nerve terminal. Clustering and fusion of synaptic vesicles. These are aligned with the mouth of the postjunctional folds  fast transmission. Signals within the synaptic basal lamina: Axons of motor neurons are capable of regenerating after being cut. In early studies, it was discussed that the nerve terminal re-grows precisely to the original synaptic site. This can occur even if the muscle cell has degenerated, leaving only its basal lamina “ghost”. In fact, the basal lamina can direct the growth of the nerve terminal even if it has been delayed (i.e., by re-cutting).

So, there must be a factor that induces AchR clustering Next slide: Destroy the motor unit. B. Muscle cell division  new myofibers. C. AchR clusters form on the regenerated muscle fibers directly beneath the synaptic portion of the basal lamina.

Therefore, the signal comes from the basal lamina and it is maintained for some time. The basal lamina also contains signals capable of directing the segmentation of the myofiber (in the absence of the nerve terminal).

Signals from basal lamina

AchR clustering on muscle fibers is induced by contact with spinal neurites (next slide) α-bungarotoxin binds (labels) AchRs. Soon after the spinal neurite grew across the muscle surface, fluorescent α-bungarotoxin appeared at the contact site => that AchR aggregation is induced.

What Signals Induce Post-synaptic Differentiation? Torpedo electric organ – a tissue homolog to muscle, but with denser innervation: - agrin – present in synaptic basal lamina. - originally deposited there during development by motor neuron terminal. Function of agrin: - Induction of Ach receptor clusters. - Regulation of distribution of other synaptic proteins (e.g., Ach esterase).

2 pieces of scientific evidence: Abs against agrin block AchR clustering. Mice lacking agrin lack normal synapses.

How does Agrin Signal? Through a receptor tyr kinase known as MuSK (muscle-specific kinase). Concentrated in post-synaptic membrane. Necessary for agrin-induced AchR clustering. Therefore, 2-way signaling must be accessing: Agrin from the nerve terminal Another signal following MuSK activation, telling the nerve terminal to settle there and differentiate pre-synaptic specializations.

Agrin-mediated signaling Motor neurons synthesize and release Agrin into the synaptic basal lamina, where it acts to maintain AChRs (green/yellow) at synaptic sites. Agrin stimulates the clustering of synaptic proteins including AChR, AChE, Rapsyn, Utrophin, neuregulin1, NRG receptors. Before innervation, AChRs (green) are spread diffusely over the surface of the myotube. Release of agrin after innervation results in the redistribution of previously unclustered AChRs to synaptic sites, adjacent to the nerve terminal.

Neural Agrin Induces AchR Clusters (next 2 slides) The chick anti-agrin blocked AchR clustering when the motor neuron was from chick, but not when the muscle cells were from chick.

Agrin induces AchR Phosphorylation prior to Clustering (next 2 slides) Agrin receptor: MuSK When cultures are exposed to agrin, MuSK is phosphorylated within minutes. AchRs are maximally phosphorylated within hrs. Receptor aggregation persists over then next few hrs, but the levels of phosphorylation declines earlier.

Agrin binds to a receptor complex and MuSK is required for Clustering (next 2 slides) Agrin activates a receptor complex involving MuSK and MASC (some accessory protein?). Rapsyn (intracellular peripheral membrane protein) required for the agrin-mediated MuSK activation to  phosphorylate and cluster AchRs. Laminin and α-dystroglycan provide additional agrin-binding sites.

Synapse at the neuromuscular junction

How are the Genes for Synaptic Specialization Regulated? Synapse-specific transcriptional control (specific for nuclei around the synapse). Signal continued in synaptic basal lamina. - Candidate: e.g., neuregulin gene products  can activate AchR gene expression. - Neuregulin acts at EGF-type receptors and may participate in the transcription of AchR genes in nuclei around the synaptic membrane. Synthezied by motor neurons. Agrin may act to localize neuregulin to synaptic site (observed to cluster along with several other proteins).

The Nectin-Afadin Adhesion System in Synaptogenesis in Hippocampal Pyramidal Neurons (next slide) Nectin-afadin system organizes adherens junctions cooperatively with the cadherin-catenin system in hippocampal pyramidal neurons. Nectin: an IgG-like adhesion molecule. Afadin: an actin-filament binding protein that connects nectin to the actin cytoskeleton. During development, nectin-1 and -3 localize at both puncta adherentia junctions and mechanically anchor at synaptic junctions. Note that the nectin-afadin and the cadherin-catenin systems co-localize around the active site zones throughout development.

Protein Dynamics During Synaptogenesis A model for the regulation of dendritic spine development. Dendrites send out long thin Processes (filopodia) that seek out and form synapses with nearby axons. Formation of filopodia is stimulated by high levels of synaptic activity (e.g. tetanus or other LTP-inducing stimuli) or by profound inactivity, presumably in conjunction with local secreted factors. Postsynaptic PDZ proteins are critical for the development of filopodia into mature spines representative mature mushroom-shaped spine is shown). The maintenance of mature spines depends on low level stimulation of the AMPA receptor.

Signals that differentiate growth cone signals into presynaptic boutons Next slide

 Trans-synaptic protein interactions implicated in synaptic contact/adhesion & synapse development: Some players Homophilic interactions: The carboxy-terminal cytoplasmic tails of -neurexin, neuroligin, EphB2, ephrinB and SynCAM (synaptic cell-adhesion molecule) bind to specific PDZ and ZO-1-domain-containing proteins, which can assemble large protein complexes that are associated with the cell-surface membrane protein. Zheng Li & Morgan Sheng 2003

Presynaptic and postsynaptic elements at glutamatergic synapses. The PDZ-containing protein PSD-95 binds to NMDA receptors, PICK-1 and GRIP, bind to AMPA receptors. Presynaptic β–neurexin binds to postsynaptic Neuroligin which is associated with NMDA receptors via PSD-95 to align pre- and postsynaptic sites. Pre-synaptic ephrinB binds to postsynaptic EphB2 receptors, clustering NMDA receptors. EphB2 receptors bind to PICK-1 & GRIP linking NMDA and AMPA receptors. Interactions between Narp & AMPA receptors have been established by in vitro binding and immunoprecipitation experiments; the importance remains to be determined.

Rat hippocampal neuron in culture expressing beta-Gal to visualize the dendrites, and immunostained for beta-Gal (green) and PSD-95 (red), a protein enriched in postsynaptic structures, the dendritic spines.