Transcript 7Synapse Form

Synapse Formation
(Synaptogenesis)
e.g., Neuromuscular Junction
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 AchRevoked 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 recutting).
So, there must be a factor that induces
AchR clustering
Next slide:
A. 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:
1. Abs against agrin block AchR clustering.
2. 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:
1. Agrin from the nerve terminal
2. 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 cadherincatenin 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 LTPinducing 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 mushroomshaped 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.