Synaptogenesis

axon guidance to synapse formation steps

1. axon guidance and dendritic growth

2. initial contact and target selection

3. recruitment of presynaptic and postsyaptic proteins

4. spine morphogenesis and maturation

DISCRETE STEPS required for maturation

Types of synapses

• chemical interneuronal

• electrical interneuronal

• neuromuscular

motor neuron axons

extend from CNS to peripheral muscles

How to label NMJ synapse

• secretes ACh

• bungarotoxin binds to AChRs

• bungarotoxin labels NMJ synapse

monoinnervation

single skeletal muscle fiber is innervated by only one motor nerve terminal

What drives AChR clustering on NMJ?

• contact dependent —> growth cone of motor neuron travels along muscle cell to drive clustering

• ECM- proteoglycan agrin

thin, specialized layer of extracellular matrix that acts as a scaffold for epithelial cells and other cell types

basal lamina

basal lamina components

• collagen

• laminin

How to show agrin is molecule that promotes AChR clustering?

• muscle cell and agrin in dish —> observe spatial AChR

• Agrin mutants lack NMJ synapses

How does agrin cause clustering?

• Agrin binds to Lrp4 receptor

• activates MuSK (receptor tyrosine kinase)

• phosphorylates AChR causing clustering

What causes dispersion of AChR

release of ACh

muscle weakness due to production of antibodies that attach AChR, MuSK, or DOk7

myasthenia gravis

tiny potentials that must be summed

axo-dendritic

strong potentials that are often inhibitory

axo-somatic

receptor types

• AMPAR

• NMDAR

AMPAR

• fast excitatory synaptic transmission

• subunits GluR1-4

NMDAR

heterotetramer- NR1/NR2A, 2B (Glu/Gly binding)

glutamatergic pre-synapse

• dock

• recruit Ca²⁺ channels

• position synapse with cell adhesion

glutamatergic post synapse

• receptors (AMPA, NMDA)

• Transmembrane organizing proteins (interact witih pre synaptic proteins to help synapses form)

• Scaffold proteins (PSD 95, hold receptors together)

• Cytoskeleton

induce receptor clustering in CNS

• Eph(post)/Ephrin(pre)

• beta-neurexin(pre)/neuroligin(post)

How did they show EphB signaling promotes glutamate receptor aggregation

• Added solution of EphrinB1 —> colocalization of NMDAR and EphrinB1

  • EphrinB1 induced NMDAR clusters on cortical neuron dendrites

• Ephrin B1/2/3 knockout —> no co-localization and no AMPAR clustering

excitatory versus inhibitory neuroligins

• excitatory: NL1

• inhibitory: NL2

molecular mechanisms holding receptors together

scaffold proteins

supports glycine/GABA (inhibitory) receptor clustering

gephyrin

How did they show gephyrin supports glycine receptor clustering

• gephyrin antisense oligonucleotide to knockdown

• lose clustering of glycine receptors

What happens when gephyrin (scaffold protein) is not scaffolded?

• impaired GABAergic synapse maintenence

• reduced association of GABAR and NL2

• epileptic

Tripartite synapse

• pre synapse

• post synapse

• astrocyte

astrocyte proteins that help with synapse formation

TSPs and Hevin

How is the pre-synapse marked

vesicle or NT synthesis enzyme

What is the PSD composed of

• receptors (AMPA, NMDA)

• scaffold proteins (PSD95, Shank, Homer)

• cytoskeleton (actin)

• transmembrane proteins (Eph, neuroligin)