Lecture 5 + 6 (Synapse formation)

What was observed in Agrin mutant mice?

-Less clustering of AChR and longer, unorganized axon terminals -Deficits of ultrastructure of synapse (fewer vesicles and lack of postsynaptic membrane thickening)

Ca2+ binds to _____ cause it to engage _____

Synaptotagmin; SNAREs

What are the two specific SNARE molecules and where are they located?

Syntaxin and SNAP25 Located on presynaptic membrane

Agrin binds to ____ which is required for ____

MuSK; receptor clustering

What is noticed when muscle fibers are damaged/motor axons are cut?

Muscle fibers degenerate and leave basal lamina ghost which contains extracellular matrix molecules that informs regenerating axon terminal where to grow/establish new synapse (reinnervate)

What do oligodendrocytes do?

Myelinate neighboring axons in the CNS Can deter axonal regeneration in CNS

____ from postsynaptic muscle cell used to test attraction of growth cone and formation of a synapse

Myoballs

What molecules are involved in synapse differentiation at neuromuscular junction (NMJ)

N-CAM 120 erb B2-4 Integrin Ropsyn ACh-R Utrophin Dystroglycon Musk

What protein modulates the selective expression of ACh receptors in subsynaptic nuclei?

Neuregulin (NRG) or ARIA

What are inductive factors that need to assemble for the function of a synapse?

Neurexin and neuroligin = post synaptic density proteins

What type of synapse is the better model and why?

Neuromuscular synapses due to their accessibility

What is the difference between interneuronal and neuromuscular synapses?

Neuromuscular: Muscle fiber contains junctional folds with ACh receptors

What did dye FM4-64 staining reveal?

New synaptic site that contains protein Bassoon

Looking at the ultrastructure of synapses, what was noticed about vesicles in the presynaptic terminal and in rodent cortex?

Newly formed synapses have few vesicles in presynaptic terminal Number of vesicles increased in rodent cortex (almost threefold) in first post natal month

What is the relationship between synapse formation and time in the visual cortex?

Number of synapses increase with time

NCAM homolog ___ in drosophila is involved in ___

FasII; target selection and synapse formation

What are the most common spine morphologies?

Filopodia, stubby, thin, mushroom

What distinguishes neural development from other tissues?

Forming functional connection between nerve cells

How do electrical synapses work?

Gap junctions between pre/post synaptic membranes permit current to flow through intercellular channels. Current flow changes membrane potential, initiating action potentials

What happens with a lack of FMRP?

Abnormal dendritic spine structure and number on layer V pyramidal cells in cortex

What happens when you inhibit MAPK?

Abolishes neuregulin-induced AChR subunit expression

How are you able to differentiate between presynaptic and postsynaptic synapses?

Anatomically by their ultra structural components

How do intracellular properties change in synapses during development? How did they test this?

As an animal grows, the mediation of the action potential shifts from being Ca2+ dependent to being Na2+ dependent Tested by recordings of spinal cord neurons when a depolarizing injection occurred: tested in neural tube, tailbud embryo, and young larve

What was observed in maturation of excitatory synapses in rat neocortex?

As brain matures, synapses become faster and sharper and more consistent effect on target neuron with time

What are the roles of glia in synapse development and function?

Astrocyte Oligodendrocyte Schwann cells Microglia Glia cells provide myelin

How do you create a biological sensor ACh ("sniffer")?

Create by excising patch of membrane from muscle cell because membrane contains ACh receptors

How do chemical synapses work?

Current flows only in response to secretion of neurotransmitter which open/close ion channels by binding to receptor molecules -No intercellular continuity --> no direct flow of current from pre to post

Why is receptor clustering at post synaptic side important?

It is a hallmark of synaptogenesis in neuromuscular junction in CNS AChR cluster on muscle cell membrane before motor axon terminal arrives

What is PSD-95? What did GFP tagging reveal in the midbrain neurons of zebra fish and xenopus embryos?

PSD=95 = postsynaptic marker protein Showed that most recent postsynaptic structures were first contacted by axonal growth cones

What do microglia do?

Participate in synapse pruning (elimination) Engulf synaptic material during postnatal development

How are we able to visualize the release of vesicles from growth cones?

Place neurons in FM4-64 (dye that does not cross membrane) When neuron depolarizes, dye enters/fuses in vesicle Dye is released when vesicle fuses with membrane

What does BDNF do (in terms of synapses)?

enhances RGC axon arbor complexity and synaptic number in vivo

How can you measure synapse number/when new synapses form?

-Electrophysiological recordings -Dyes that reveal synapse vesicle release (FM dye)* -Dyes that reveal localized changes in calcium levels -Synaptic proteins marked with fluorophores (live imaging)* -Immunostaining for proteins (fixed tissue) -Electro microscopy analysis of fixed tissues*

What are major contributors of CNS synapse formation?

-EphrinB-Eph8: glutamate receptor expression -Neurotrophin-Trk signaing -Neuroligin-Neurexin interaction at synapse

What is agrin and its function?

-Found to be a proteoglycan that is a presynaptic molecule that induces AChR clustering in NMJ -Secreted by growth cone, induces accumulation of neurotransmitter receptors in muscle fiber and triggers synapse differentiation -Expressed in motor neuron cell bodies and muscle cells (two different isoforms)

How does the density of synaptic spine change with age in B-catenin knockout mutants?

-Increases in childhood -Decreases (not as much as normal) in adolesence -Stabilizes (still higher than normal)

How do functional connections form?

-Laminar specificity -Cellular specificity -Subcellular specificity -Synapse-type specificty

The stages of synapse formation: 3. Stabilization of synapse

-Mature synapse exhibits accumulation of vesicles, dense extracellular matrix in left and postsynaptic density

How does density of synaptic spines change with age?

-Peaks in childhood -Decreases in adolescence -Stablizes in adulthood

The stages of synapse formation: 2. assembly of synaptic machinery

-Vesicles found in both pre and post synaptic elements --> add membrane and extracellular glycoprotein -immature synapse display vesicles

The stages of synapse formation: 1. initial contact

-presynaptic growth cone comes into contact with postsynaptic membrane -Filipodia retrace and membrane become tightly joined

What is the mechanism mediating synaptic transmitter release?

1. AP induces elevation of intracellular Ca2+ 2. Increased Ca2+ triggers interaction between synaptotagmin and SNARE (syntaxin, SNAP25) --> binds two membranes together 3. Induces vesicle fusion causing release of neurotransmitter into synaptic cleft

Describe the steps in neuromuscular junction formation

1. Growth cone approaches target muscle fiber 2. Upon contact with target, pre/post synaptic terminal undergo changes 3. Accumulation of neurotransmitter receptor in post 4. Upregulation of neurotransmitter receptor 5. Competition for synaptic space and formation of junctional folds

Describe the stages in development of interneuronal synapses

1. Growth cone interacts with developing dendrite through 2-way filopodial communication 2. pre/post synaptic terminal form unspecialized, functional contact 3. Synaptic vesicles at presynaptic terminal accumulate triggering neurotransmitter release and further synaptic differentiation 4. Differentiation of presynaptic terminal follows by differentiation of post (by accumulation of components) 5. Recruitment of organizing molecules (PSD95) followed by rapid neurotransmitter receptor accumulation ad then functional maturation of synapse

What are the steps of synapse formation/maturation?

1. Initial contact 2. Assembly of synaptic machinery 3. Stabilization of synapse 4. Synapse do not function in mature manner for a while after they are formed

What do you notice in mutant mice in which nerve fails to dvelop?

AChR receptors are in muscle fiber but not clustered

How does spine size differ between control spines and B-catenin overexpressed spines

B-catenin overexpressed spines are relatively larger in size and range than control spines

Dendritic spines comete for ____ to ____

B-catenin; survive

How are synapses classified?

By either chemical or electrical then by type of chemical signal (neurotransmitter) used

How do Schwann cells and oligodendrocytes form myelin?

By progressively wrapping sheets of their cytoplasm around the axon

How was the protein Bassoon discovered?

By staining with antibodies revealed by staining

____ participate as signals to induce pre and postsynaptic site differentiation

CAMs

What regulates growth cones stopping when they find target?

Ca2+ levels regulate this mechanism Levels rise when touching muscle cell

Describe control beads vs cad-coated bread in terms of percentages of time neighboring spine disappears and spine size

Cad-coated bead higher in percentage compared to control Spine size increases with time in coated beads and decreases in time with control beads

What are adhesive factors that need to assemble for the function of a synapse?

Cadherins and protocadherins

What does sclerosis depend on? How does this affect signaling?

Depends on balance of demyelination and remyelination Signal from the brain may be blocked if there is damage myelin

Intracellular signaling induces...

Differentiation of newly formed synapses from moment of contact

What is the distributed circuit model?

Each presynaptic neuron contacts many postsynaptic neurons and makes contact with each of them (spines)

____ contains a factor that induces AChR clustering Describe the frog experiment

Extracellular matrix Motor axons were cut leaving only basal lamina which contains extracellular matrix molecules. New myofibers are generated as result of cell division AChR clusters form on regenerated muscle fiber beneath basal lamina

T/F: Morphology can tell us about function

F

What is fragile X syndrome (FXS)? What happens when you increase proteins in FXS?

FMR1 protein (coded by gene fro fragile X) represses translation from a number of proteins (PSD-95, potassium channels, etc) Overabundance FXS = increase synapse number

Changes in chloride channels shift GABA receptors from excitatory to inhibitory

High intracellular Cl- opens channel allowing the Cl- to exit (depolarizing) When potassium-chloride transporter reduces intracellular Cl- opening channel this hyperpolarizes cell

What do neuroligin-coated beads do?

Induce cultured neurons to produce presynaptic protein synaptotagmin -Presynaptic protein synapsin accumulates only in response to beads coated with NLG

What does EphrinB1 and EphB2 receptors do?

Induce neurotranmitter receptor clustering in CNS

What molecules are extrasynaptic?

Laminin Collagen

What molecules are synaptic?

Laminin-5 Aria Agrin Acetylcholinesterase Collagen IV

Why do synapses form around soma/cell body? Lesson?

Lesson: location of synapse is important Important for firing action potential or halting activity

When did major vertebrate synaptic evolution duplication/divergence occur?

Many synaptic-related genes were duplicated/diverged ~ 500 years ago

What are membrane-associated guanylate kinases (MAGUKs)?

Molecules expressed in excitatory synapses that regulate clustering

What is the relation between synaptogenesis and cortical areas?

Most synapses formed after birth and peak synaptogenesis varies caries in different cortical area

What resulted in the evolution of synapses?

Proliferation of receptor subunits allowing more varied and complex synaptic signaling

What is Gephyrin?

Protein that is required for clustering of some GABA receptors

What molecular mechanisms hold the clusters of receptors together in CNS synapses?

Proteins built into cytoplasmic surface that bind both membrane receptors and cytoskeletal elements

What do Schwann cells do?

Provide layers of myelin for axons in PNS and individual axons outside nerves

What is the difference between reticular network vs neuron theory?

Reticular network: thought to be syncytium of cells joined by their processes --> allows electrical activity to travel either direction Neuron theory (separate): recognized that chemical synapse would permit electrical activity to travel only one direction (Charles Sherrington)

What was revealed with staining with PSD-95 antibodies?

Revealed differentiated postsynaptic sites (on dendrites) that is absent in newly formed synapse

Describe the steps in the development of muscle fibers

Satellite cells --> dividing myoblasts --> initial myotube formation --> myotube maturation --> muscle fiber

What are interneuron synapses?

Synapses formed between neurons

What happens if you block translation of Gephyrin?

The receptors do not form clusters in neuronal membrane

What did Golgi stain spinal cord tissues show about axonal growth cones and dendritic growth cones?

These two growth cones grow towards each other Synapses are dynamics

Where are excitatory synapses made in the cortex?

They are made on dendritic spines on pyramidal neurons in the cortex

What are the two types of ion channels? How does this effect development?

Voltage gated channels and ligand gated channels Channels can change course of development altering its neurophysiological properties

What happens when the "sniffer" is close to the growth cone? What does this indicate?

When sniffer pipette is put close to growth cone, activity increases (downward deflection) indicating growth cone is releasing ACh

How can vesicular release occur in mammalian CNS?

by growth cone filopodia

CAMs near synapse participate in...

early synaptic partner recognition and later in stabilization

Neuregulin is important for ____ How many isoforms from 1 NRG1 gene? Receptors are called ___ of receptor ____ Needs to colocalize with ____ receptors at NMJ NRG stimulates phosphorylation of ___ and ___

migration and glial differentiation 20 EGF family; tyrosine kinases (erbBs) at least 3 erbB receptors; mitogen-activated protein kinases (MAPK)

In order to measure neurotransmitter release, use ____ as a probe to record spontaneous activity ____ Results:

monocyte (muscle cell); post synaptic currents PSCs Spontaneous PSCs show as downward deflections, response requires both presynaptic (transmitter) and post synaptic (receptor) components

Synapses mature ____ during development

morphologically

Growth cones contain ____ mechanism which was demonstrated in ____ After the first two days of culture, spinal neurons produced growth cones...

rudimentary neurotransmitter- releasing; Xenopus spinal neurons (motor neurons) extend neurites and form functional cholinergic synapses with neighboring muscle cells