Synapse Formation II April 20, 2007 Mu-ming Poo 1. Activity-dependent synapse maturation 2.Activity-dependent regulation of muscle fibers 3.Polyneuronal innervation and synapse elimination 4.Denervation and Reinnervation of muscle fibers -- Denervation effects in muscle -- Specificity in muscle reinnervation 5.Specificity of neuromuscular synapse formation during development 6.Formation of central synapses

Innervation-induced changes in AChR Properties Embryonic AChRs: -- low conductance and long opening time (pentamer consisting of  subunits), resulting in slow synaptic current -- mobile (diffusely distributed), short half-life (1 day) Mature AChRs: -- high conductance and short opening time (pentamer consisting 2  subunits, resulting fast synaptic currents -- stable (clustered), long half-life (5 days)

Summary on the Role of Synaptic (Electrical) Activity on Synapse formation 1. Initial clustering of AChR activity-independent (by unknown factor) 2. Activity-dependent processes at developing synapses -- Increased AChR lifetime (from 1 day to 1 week) -- Down-regulation of extrasynaptic AChRs -- Maturation of AChR clusters (pretzel-shaped) -- Switch of AChR subunit from α 2 βγδ to α 2 βεδ (change in kinetics) -- Synaptic competition and elimination of polyneuronal innervation 3. Activity-dependent mechanisms -- Factors secreted as a result of activity? -- Depolarization-induced Ca 2+ influx and downstream effectors.

Innervation-dependent muscle contractile properties -- Embryonic: mostly slow (S) motor units -- Mature: more fast (FF) motor units -- The transition is induced by innervation, change in expression of different types of myosin isoforms (which different ATPase activities) -- Cross-innervation of the muscle (in animal experiments) results in switching of muscle properties according to the nerve type. -- Altering the pattern of activity in the nerve has similar effects, i.e., applying phasic stimulation (transient high-frequency bursts) change the slow muscle into fast muscle

Elimination of Polyneuronal Innervation at NMJs Each muscle fiber innervated by several motor axons at birth 1.Establishment of several inputs results in refractory of muscle (extrasynaptic) surface to further innervation 2.Within 2 postnatal weeks, all but one motor axon remains 3.Competition of postsynaptic territory occurs at the endplate among several terminals. Synapse becomes weakened as it looses the territory, and eventually retracts. 4.Competition mediated by muscle, two inputs can compete even separated by up to 1 mm. 5.Competition activity dependent – blockade of synaptic activity and excessive stimulation slow down and speed up elimination, respectively

Green - motor neuron. Red – AChR (rhodamine tagged α– BTX) Innervation of individual neuromuscular junctions by axonal branch trimming Each muscle fiber change from poly to single innervation Number of fibers innervated by each motor neuron is reduced This is mediated by retraction of axonal branches

Synapse elimination: activity-dependent competition Competition Remove some motor neurons, all muscle fibers are still innervated; remove the original innervation, other motor neurons take over Activity-dependent TTX (blocks Na channels, action potentials) blocks elimination (the same muscle fiber remain innervated by multiple motor neurons)  -bungarotoxin (blocks AChR) blocks elimination

Mechanism of Synaptic Competition 1.Trophic factor hypothesis: Competition for muscle derived maintenance factor (“synaptotrophin”), and the active nerve terminal takes most of the factor and becomes stabilized, while inactive terminals are eliminated due to the lack of the factor. (Secreted factors added to the NMJs can retard elimination: BDNF, leukemia inhibitory factor, CNTF, FGF-2, GDNF, but in vivo evidence of secretion of these factors lacking) pre- post-

Mechanism of Synaptic Competition 2.Toxic factor hypothesis: Elimination due to muscle-derived toxic substance, and active nerve terminal becomes immune. -- Protease inhibitor slows down elimination process -- Calcium activated neutral protease destablized nerve terminal -- Thrombin, a serine protease, speeds up elimination -- Agrin can act as protease inhibitor 3. Retrograde factor hypothesis: Intracellular factors (“synaptomedins”) in the muscle cell triggered by active nerve terminal selective stablizes and destabilizes active and inactive nerve teriminals, respectively, through transmembrane actions on the nerve terminals -- Levels of AChRs and rapsin begin to decline at the synaptic site before nerve terminal withdrawal.

Ann. Rev. Neurosci. (1999)

Denervation and Reinnervation of Muscle

Effects of denervation in skeletal muscles 1.Paralysis (immediately): loss of sensation and motility 2.Fasiculation (immediately): spontaneous firing of the injured axon, causing twitching of motor units. 3.Fibrillation (days) : spontaneous twitching of individual muscle fibers due changes in muscle excitability (e.g., Na channels) 4.Supersensitivity to ACh (days): increased AChRs in the muscle membrane, due to elevated extrasynaptic AChRs (increased AChR synthesis). 5.Muscle atrophy (>1 week): loss of muscle proteins 6.Receptiveness to innervation: allows reinnervation of the muscle by motor axons

Regeneration of NMJs after denervation 1.Regenerating motor nerve may be guided by remaining perineurial tube to the original endplate to form synapse 2.Ectopic synaptic sites may be formed by reinnervating axons. (Denervated muscle becomes hypersensitive to ACh, and loses refractoriness to innervation) 3.Reinnervation is NOT muscle-specific -- uncontrolled facial expression -- “crocodile tear syndrome” (Spontaneous tearing during normal salivation of eating, due to mis-innervation of the tear gland by axons destined for salivary gland following facial nerve damage) -- importance of intrafascicular nerve repair after peripheral nerve transection