Development of Nervous System PDF

9.2 Synaptic modules Presynaptic – Vesicle fusion – Active zone Postsynaptic density (PSD) Figure 149. Pre and post synaptic modules 9.3 Presynaptic assembly 9.3.1 Vesicle fusion SNAREs: Synaptobrevin = vesicular SNARE...

9.2 Synaptic modules Presynaptic – Vesicle fusion – Active zone Postsynaptic density (PSD) Figure 149. Pre and post synaptic modules 9.3 Presynaptic assembly 9.3.1 Vesicle fusion SNAREs: Synaptobrevin = vesicular SNARE protein = v-SNARE Syntaxin = target SNARE protein = t-SNARE -> corresponding to Synaptobrevin SNAP-25 -> zipper bringing together v- and t-SNARE in close proximity The zipped up complex allows the vesicle and membrane to fuse Figure 150. SNAREs FRET: = Förster/Fluorescence Resonance Energy Transfer The energy transfer depends on the distance of the two floor forces It works if the floors are close together, but does not if the floors are apart more than 6-8nm The donor is excited and the energy is transferred to the acceptor, which makes the donor a little dimmer -> can be observed 90  Figure 151. FRET experiment to prove test the SNARE complex Figure 152. Probing intermolecular distance with FRET Donors and acceptors plus SNAP-25 and t-SNARE were added to Lipososomes Synaptic vesicles were purified and mixed with the liposomes If the synaptic vesicles fuse with the liposome, the energy would decrease upon fusion Vesicle fusion can be reconstituted in vitro, in the absence of active zone components or Ca2+ Fusion with only SNARE-complexes is a slow process: ca 40’ To fire fast, Ca-influx is necessary 91  Figure 153. FRET in Liposomes 9.4 The active zone assembly Active Zone (AZ): Platform for rapid fusion of synaptic vesicles after Ca2+ influx AZ-membrane is decorated by a proteinacious cytomatrix (a set of specialized proteins) Questions Answers to the questions based on several experiments: Questions: Contact with postsynaptic partner required? Delivery as preassembled units or single components? Are “orphan” active zones functional? Answers: Co-culturing of neurons with HEK-cells, which express Neuroligin (-> Neurons form synapses with the HEK- cells): Presynaptic differentiation can be induced by expression of a single postsynaptic cell adhesion molecule (Neuroligin) on non-neuronal cells! Injection of polylysine-coated beads into the cortex: Active-zone formation after contact with polylysine-coated beads! Staining for synaptic vesicles, dendrites and AZ-protein: Active-zone assembly does not require a postsynaptic partner (self-assembly of active zones)! Probing Exocytosis using FM (= Fei Mao-43) Destaining: – Fluorescence signal occurs if the dye molecule hits a charged (polar solvent: H20) membrane! – “Orphan synapses” can undergo exocytosis! Partial axonal co-transport of active zone proteins and synaptic vesicles in presynaptic lysosome-related vesicles (PLVs) requires Arl8 Microtubule (MT) polarity and specific MT-associated proteins specifically target synaptic components to synapses in axons or dendrites 9.4.1 Genes involved in Active Zone Assembly in Drosophila Synapse Defective-1 is required for normal synaptic vesicle targeting in the Drosophila PNS and CNS, as well as in the mouse CNS 92  The small GTPase Rab3 is required for normal AZ distribution, and pre/post matching No large defect in synaptic transmission at rab3 mutant synapses Figure 154. Presynaptic Development of the AT THE DROSOPHILA Figure 155. Presynaptic Development in C. elegans, Drosophila and mammals 93 Figure 156. Core acitve zone proteins 94 Figure 157. Active zone assembly 95 Figure 158. Branch specific axonal synaptogenesis: The phosphatase Prl-1 specifies subcellular synapse location and axon Prl-1 Phosphatase branching of regenerating in an axon liver branch specific fashion in the Drosophila CNS 9.5 Post-synaptic density (PSD) Neurexin expression in non-neuronal cells clusters glutamate- and GABA postsynaptic scaffolding proteins in dendrites Neurexin expression in non-neuronal cells induces accumulation of postsynaptic scaffolding proteins in dendrites Neuroligin expression in non-neuronal cells clusters glutamate- and GABA-containing synaptic vesicles Neuroligin isoforms are involved in determining the sign of a synapse. Questions with NL1,3 et NL2 Figure 159. Postsynaptic density and active zone assembly 96  Figure 160. Excitatory vs. inhibitory synapse 9.5.1 Synaptic CAMs Synaptic CAMs (= Cell adhesion molecule) inducing pre- or postsynaptic formation. Questions with proteins expressed Figure 161. CAM-specific induction of presynaptic and postsynaptic compartment 9.5.2 PSD assembly ‘Nonsynaptic’ clusters of postsynaptic scaffolding proteins (-> co-transport of postsynaptic proteins)? Contact per se is not sufficient to drive synapse formation -> scaffolding proteins Mostly, sites apposed to stationary nonsynaptic scaffold clusters are readily transformed to active presynaptic terminals AMPARs and NMDARs are shipped in different vesicles. PSD-95 and SAP-90 are recruited to synapses from cytoplasmic pools 97  Question Figure 162. Non-synaptic clusters of postsynaptic scaffolding proteins 9.5.3 Glutamate Uncaging Experiment 2-Photon glutamate uncaging can be employed to induce neurotransmitter release at individual synapses Glutamate uncaging can be used to “grow” a postsynaptic structure (spine) layer 2/3 within pyramidal seconds -> Spinogenesis can be rapidly induced by presynaptic activity! Neuroligin-1 is required for glutamate uncaging induced spinogenesis Clusters of the presynaptic protein Munc13 likely represent presynaptic release sites Glutamate uncaging induces spine growth and accumulation of the postsynaptic density protein PSD-95 in mature synapses -> Plasticity! Experiment Figure 163. Glutamate uncaging Spinogenesis without neurotransmitter release: Normal spine development in Munc13-1/Munc13-2 double-KO mice without neurotransmitter release Normal glutamate uncaging-induced spine development in Munc13-1/Munc13-2 double-KO mice Glutamate uncaging-induced AMPAR recruitment in Munc13-1/Munc13-2 double-KO mice 98  Figure 164. Spinogenesis in control group compared to Munc13-1/-2 double KO mice 9.6 Postsynaptic and transsynaptic molecules Excitatory synapses are characterized by specific pre- and postsynaptic proteins Inhibitory synapses are characterized by specific pre- and postsynaptic proteins Figure 165. Postsynaptic and transsynaptic molecules 9.6.1 Transsynaptic molecules Homophilic and heterophilic interaction between cell adhesion molecules at synapses. Postsynaptic GluRδ2 interacts with presynaptic neurexins in the presence of “cerebellin 1 precursor protein” (Cbln1) Incubation with exogenous Cbln1 for 7 days restores the reduction in VGLUT1 signal (= Presynapse) in cbln1 KO mice Cell adhesion molecules play specific roles in synapse formation -> “CAM code” is involved in determining synaptic specificity Transsynaptic cell adhesion molecules play a role in synapse formation, and in synaptic plasticity at mature synapses Cell adhesion molecules have been linked to autism-spectrum disorders 99 Figure 166. Transsynaptic Molecules – Example Parallel Fibre Figure 167. Extracellular matrix (ECM) molecules 100  Figure 168. Transsynaptic molecules in the process of synapse formation and plasticity 9.6.2 ————— Modules Synaptic molecules PALM: Photoactivated localization microscopy PALM is a “super-resolution” light microscopy approach that is based on stochastic activation of fluorophores to increase spatial resolution PALM can be used to estimate PSD-95 molecule number Figure 169. oben: PALM / unten: Stochastic activation of fluorophores and localization of individual molecules Pre-/post Alignement (Nanocolumns): PALM reveals aligned “nanoclusters” of presynaptic and postsynaptic molecules (“nanocolumns”). 101  Figure 170. Nanocolumns 9.6.3 Timing of Synaptogenesis Cell adhesion molecule interaction => presynaptic assembly => postsynaptic assembly (with exceptions) Figure 171. Timing of Synaptogenesis 9.7 Maturation: Elimination/competition Differences between synapses: Synaptic (transmission) strength: Degree of postsynaptic voltage/current change in response to action-potential stimulation 102  Figure 172. Synapse transmission strength 9.7.1 Maturation Neurotransmitter release probability depends on the number of synapses that are made onto a dendritic branch Neurotransmitter release probability (and thus synaptic strength) depends on the number of synapses per dendritic branch Many synapses are eliminated during maturation: Elimination of multiple innervations during maturation Synapse elimination results in monosynaptic innervation 103  Figure 173. Maturation: Elimination of multiple innervation and the result of synapse elimination (monosynaptic innervation) 9.7.2 Competition Input segregation precedes terminal withdrawal from the NMJ All synapses but one are eliminated during development of climbing fiber-cerebellar Purkinje synapses 104 Figure 174. oben links: Input segregation precedes terminal withdrawal from the NMJ / unten links: Patterns of motoneuron activity modulate synapse elimination at the NMJ / oben rechts: All synapses but one are eliminated during development of climbing fiber- cerebellar Purkinje synapses Figure 175. Activity-dependent synapse elimination of cerebellar climbing fiberPurkinje cell synapses requires PQ-type Ca 2+ channels and Arc 105 9.8 Plasticity 9.8.1 Climbing fibers Only the “largest” climbing fiber input becomes stronger! Figure 176. Climbing fibers 9.8.2 Synaptic plasticity and competition Synchronous activation of climbing fiber (CF) and purkinje cell (PC) is commonly used to induce long-term potentiation (LTP) Long-term potentiation can be only induced at large inputs through an increase in conductance of AMPA-type glutamate receptors Long-term potentiation only occurs at large inputs, and results in increased conductance of AMPA-type glu- tamate receptors The “winner synapse” undergoes LTP (becomes stronger), and eliminates the other synapses through postsy- naptic Ca2+ /Arc signaling 106 Figure 177. oben links: synchronous activation of CF and PC / unten links: LTP induction / oben rechts: Winner synapse / unten rechts: LTP occuration 9.9 How to build a synapse? Transsynaptic signaling through cell adhesion molecules plays key roles in synaptic development and -plasticity Evidence for synaptic activity-dependent and -independent synapse formation Assembly of presynapse => assembly of postsynapse Maturation (often) involves synapse elimination, which is activity dependent Synaptic plasticity of mature synapses can involve mechanisms that were used during synaptogenesis 10 Adult neural stem cells 10.1 Analysis of adult neurogenesis 10.1.1 BrdU labelling BrdU labeling in mice: BrdU is incorporated in the newly synthesizing DNA -> red label The splicing factor NeuN is exclusively expressed in neurons -> green label 107