BIPN140 Lecture 13: Synapse Formation (Synaptogenesis) 1. Neuromuscular Junction (NMJ) Development 2. Synaptogenesis at Central Synapses Su (FA16) Ultrastructural Image of an NMJ Active Zone Basal Lamina Junctional fold Active zone Neuromuscular Junction (NMJ): 1. A model system for study of synapse components and formation. 2. Characteristic arrangement of cell types, pre- and postsynaptic components, and specialized basal lamina. 3. Morphological features of NMJ appear sequentially; differentiation of motor neuron (MN) and target synaptic specialization appear coordinated. 4. Initial stages are activity independent, however, maturation requires activity. 5. Synaptic location is random, yet organization is stereotypic. 6. Morphological features of a mature NMJ: (1) Pre- and postsynaptic membranes are separated by a synaptic cleft that contains basal lamina (organized sheets of extracellular matrix components) and extracellular matrix proteins. (2) Vesicles are clustered at presynaptic release sites (active zones), transmitter receptors are clustered in the postsynaptic membrane. (3) Nerve terminals are coated by Schwann cell processes. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-7)
NMJ Develops in Sequential Stages (Fig. 23.11) 1. Growth cone approaches Prior to contact, MN can already release ACh. AChRs are already expressed on muscle membrane. 2. Upon MN contact with muscle, existing AChRs show dramatic clustering and MN form vesiclerich terminal arborizations. 3. Basal lamina starts to form. Upregulation of synaptic nachrs and down regulation of extrasynaptic or extrajunctional nachrs. 4. Multiple axons converge onto the same muscle fiber. MNs and muscles are primed for function. Upon contact synaptic transmission commences, though with low efficacy. Both pre- and postsynaptic components are already present prior to contact. Many of the interactions are modulatory in nature, insuring matching of pre- and postsynaptic components. Initial events are organizing instead of instructive. Immature: Multiple innervation Mature: Monosynaptic innervation 5. Only one axon remains in mature NMJ (monosynaptic, via competition) One muscle fiber is innervated by a single axon; a single axon can innervated multiple fibers (motor unit) (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-7) Post- (muscle fiber) => laminin => Presynaptic specialization Postsynaptic muscle release signaling molecules to instruct the organization of active zones in the small portion of the axon terminal that contacts the muscle surface, in particular the regions opposing the junctional folds. Denervation/re-innervation experiments: axotomy => new NMJ forms at the original sites Denervation + muscle elimination => new NMJ still forms at the original sites => components of the basal lamina organize presynaptic specialization. One such component is laminin, an extracellular matrix molecule (ECM) and the major component of all basal lamina that promotes axon outgrowth in many neuronal types. Laminins are synthesized by muscle cells and incorporated into the basal lamina. MN terminals + laminin => stop growing, accumulate SVs, and acquire the ability to release NT. Mechanism: laminins bind to VGCCs at the axon terminal, leading to the recruitment of other components of the release apparatus. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-9)
Pre- (MN terminals) => Agrin => Postsynaptic AChR clustering Upon MN contact with muscle, the following events take place. (1) Active clustering of existing AChRs (receptor redistribution: 1000 m -2 to 10,000 m -2 synaptic and10 m -2 extrasynaptic) (2) Up-regulation of synaptic AChR synthesis. (3) Repression of extrasynaptic AChR synthesis. AChR clusters Agrin, a large multi-domain proteoglycan secreted by MN into the basal lamina, induces aggregation of AChRs at synaptic sites (Agrin mutants have very few receptor clusters). Agrin signaling requires MuSK, (musclespecific trk-related receptor with a kringle domain) is a receptor tyrosine kinase expressed on muscle surface (MuSK mutants have similar phenotypes as Agrin mutants). Activated MuSK recruits Rapsyn, an AChR interacting protein, to induce AChR clustering. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-11) Roles of Agrin & ACh in NMJ Postsynaptic Development: (+) and (-) Regulations MN also secret dispersal factor to disperse spontaneously formed AChR aggregates outside synapses. ACh is the major dispersal factor; clustering persists in Agrin/ChAT double mutants. Agrin may render AChRs immune to the de-clustering effects of acetylcholine.
Pre- (MN terminals) => Neuregulin => Promote AChR synthesis Junctional nucleus neuregulin Extra-junctional nucleus A muscle fiber has multiple nuclei that can produce gene product independently. AChR transcription is enhanced in junctional nuclei: Neuregulin (a.k.a. ARIA, acetylcholine receptor inducing activity), a trophic factor secreted by MNs, stimulates AChR transcription via activating erbb receptor tyrosine kinase => ras/mapk => activate transcription. Neuregulin heterozygotes show 50% reduction in AChR density and reduced amplitude of MEPP. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-12) Pre- (MN terminals) => ACh => Suppress Extrajunctional AChR synthesis Junctional nucleus neuregulin Extra-junctional nucleus Depolarization Repression of extra-junctional AChR synthesis requires activity. Denervation or paralysis => up-regulation of AChR synthesis (activity is required for repression). Direct electrical stimulation of muscle also represses AChR transcription. Muscle depolarization => Ca 2+ influx => PKC activation => phosphorylation of transcription factors to inactivate them => shutting off AChR transcription globally. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-12)
Elimination of Multiple Innervation (Fig. 23.12) Motor neuron #1 Motor neuron #2 AChR Elimination of multiple innervation in the PNS. Live imaging of the same NMJ: At P11, two axons (blue & green) innervate the same muscle. AChR labeled in red. By P12, the proportion of territory occupied by the green and blue axons has begun to shift, with the green axon terminal expands. By P14, the blue axon has fully retreated, its synaptic terminal transformed into a large retraction bulb then fully withdrawn from the synaptic site. Some Neuromuscular Synapses are Eliminated after Birth Local blockade of AChR 1. Different axon terminals have different degrees of innervation. 2. The dominant terminal eventually stay and extend the terminals. 3. Winner likely sends loser signals to retract its terminals. 4. The loser does not die but innervate other muscle fibers. At birth, individual immature muscle fibers are innervated by multiple MNs (polyneuronal innervation). However, after two weeks each fiber is innervated by only a single MN. Why? To ensure that each muscle fiber is innervated; to allow axons to capture appropriate number of target cells. Synapse elimination is an orderly process (usually takes weeks), whereby both loser and winner maintain pre-synaptic function. However, at some point the loser rapidly changes morphology and retracts. Block AChR function locally lead to retraction of nerve terminals (activity dependent).
Summary: NMJ Development (Naguib et al., Anesthesiology 96, 202-231, 2002) Central v.s. NMJ Synaptogenesis NMJ Excitatory Central Synapse Axon Varicosity Inhibitory Central Synapse Axon Varicosity Presynaptic bouton: small axonal varicosities, ~1 m in size, establishing contacts with postsynaptic cells. Dendritic Spine Dendritic Shaft Morphological similarities => development of pre- and postsynaptic specializations. Diverse morphologies: glutamatergic synapses (spine or non-spine); GABAergic synapses (mostly on dendritic shafts where microtubules are abundant). Different types of NT receptors have different intracellular interacting proteins and/or auxiliary subunits (anchoring, trafficking, channel properties, etc.) Active zone: presynaptic region where SV fusion can occur (visible in EM as a meshwork of proteins). Postsynaptic density: opposing the active zone, clusters of NT receptors, channels, signaling molecules & scaffolding proteins. (Kandel et al., Principles of Neural Science, 5 th Edition, Fig 55-14)
Signaling Pathways Regulating CNS Synaptogenesis Axo-dendritic synapse Synaptogenesis: a multistep sequential process Initial contact: cell adhesion molecules (CAMs), such as cadherins and protocadherins function as adhesive factors. Presynaptic induction: additional CAMs (inductive factors) induce the formation of presynaptic active zones and stabilize the nascent synaptic junction. Inductive CAMs also promote the recruitment of postsynaptic glutamate receptors & scaffolding proteins. Stabilization: likely mediated by neural activity (turnover of synaptic components and synapse elimination). (Waites, Craig & Garner, Annu Rev Neurosci, 2005) Filopodia: dynamic protrusions found on axons and dendrites, particularly at growth cones Cell Adhesion Molecules Guiding Synaptic Specificity (23.4 & 8) 1. Initiation of synaptogenesis depends on local recognition between the presumptive pre- and postsynaptic membranes mediated by members of the Ca 2+ -dependent cell adhesion molecules (CAMs, they are transmembrane proteins), cadherin superfamily (a large family with great diversity, multiple genes & alternative splicing). 2. Cadherins/protocadherins: attaching pre- to post-synapse via homophilic interactions. Cadherins link to actin cytoskeleton via catenin. Protocadherins may mediate a variety of intracellular events. 3. This local recognition is accompanied by the initial accumulation of synaptic vesicles as well as transport vesicles (80 nm dense-core vesicles) that contain molecular components for the presynaptic active zones, such as t-snares (e.g. syntaxin, SNAP25) and multidomain scaffold proteins of the active zone (e.g. Bassoon, Piccolo).
Potential Molecular Mediators of Synapse Identity (Fig. 23.9) Genes encoding Pcdhs have multiple sites for alternative splicing and thus can encode a large number of variants of the same protein (allowing specificity between pre- and postsynaptic neurons and/or self recognition/avoidance). Variable extracellular domain + conserved intracellular domain => A vast cell surface diversity arises from this combinatorial expression. A synaptic zip code? Predominantly expressed in the developing nervous system. Different synaptic sites may have different complements of Pcdh molecules to confer specificity to those synaptic junctions. Inductive Factors for Synaptogenesis (Fig. 23.8) Inductive factors bring together machinery of pre- and postsynaptic sites. Once the initial specialization is established, additional adhesion molecules are recruited, including synaptic cell adhesion molecule (SynCAM), a member of the calciumindependent CAM and Ig superfamily of adhesion molecules, also via homophilic interactions, important for the formation of presynaptic boutons. Overexpression of SynCAM in cultured neuron promotes synaptic formation, in particular presynaptic differentiation. Other inductive factors: neurexin (pre) + neuroligin (post); ephrinb ligands (pre) + EphB receptor (post) Interaction between pre- and postsynaptic inductive factors turns on signaling events to initiate differentiation of the presynaptic active zone and postsynaptic density. The presynaptic terminal also releases molecules such as neuregulin (via binding to ErbB RTK) that influence the expression and clustering of postsynaptic receptors and associated proteins (like in the NMJ).
Neurexin (pre-)-neuroligin (post) Interaction Promotes Synaptic Differentiation The interaction of neurexin with neuroligin is central for recruiting and retaining cytoskeletal elements that localize SVs to the presynaptic terminal. Align pre- and postsynaptic compartments; binding leads to clustering of the complex. Cultured brain neurons + fibroblast expressing neuroligin => contacting neuronal processes develop presynaptic specializations (clustered neurexin, Ca 2+ channels and SVs). Presynaptic signaling molecule: neurexin, which associates with synaptotagmin (Ca 2+ sensor for SV release), localizing VGCC to ensure local SV release and presynaptic differentiation of active zone. Postsynaptic signaling molecule: neuroligin, interacting with PSD-95 (postsynaptic scaffolding protein for glutamatergic synapses), promoting clustering of NT receptors, formation of PSD, and aligns pre- and postsynaptic components. Ephrins and Eph Receptors (Fig. 23.4) Ephrin ligands/eph receptor tyrosine kinase: both ephrin and Eph receptors can initiate intracellular signaling events (ephrin can reverse signal ). Diverse ephrin ligands (5 ephrin-a, 3 ephrin-b) and Eph receptors (9 EphA & 5 EphB, the largest subfamily of receptor tyrosine kinases, RTKs) EphrinB (pre)+ EphB receptor (post): activation of EphB receptor leads to EphB aggregation and is important for clustering of NMDA-Rs and enhances their calcium permeability. Also important for dendritic spine development.
Developmental Sequence of Glutamatergic Synapses Axo-dendritic synapse 1. Filopodium extends from dendrite 2. Initial contact with axon terminal/branch (adhesive factors). 3. Inductive events: specific trans-synaptic acting components. 4. Maturation (asymmetric): (a) presynaptic active zones (b) postsynaptic scaffolds: clustering of receptors, segregated receptor distribution, signaling molecules, ion channels, cytoskeletal reorganization, etc. (c) increased NT release and receptor responsiveness
Fig. 1. Exposure of mice to increased circuit activity reveals an NPAS4-depdendent regulation of inhibition in vivo. Fig. 2. Behaviorally induced NPAS4 differentially regulates inhibitory synapse function across the somato-dendritic axis of pyramidal neurons.