Lecture 25- Synaptogenesis II

Question 1

Where do excitatotory synapses occur?

Answer 1

-on dendritic spines

Question 2

What is the pre-synaptic active zone?

Answer 2

• site of synaptic vesicle docking and fusion for neurotransmitter release

Question 3

What is the post-synaptic density?

Answer 3

• electron-dense specialization of the excitatory post-synaptic membrane • contains glutamate receptors, scaffolding proteins (e.g. PSD-95), adhesion & signalling molecules, cytoskeletal proteins

Question 4

How do inhibitory synapses look?

Answer 4

-Distinguish able from excitatory synapse by site of formation and “symmetrical” appearance (no obvious thickened post-synaptic density) -inhibitory synapses are on the dendritic shaft

Question 5

What are the differences between NMJ and a CNS synapse?

Answer 5

• Same basic elements (synaptic vesicles, synaptic cleft, post synaptic density) • Less elaborate (no “pretzel” and no junctional folds) • No basal lamina so pre-synaptic“bouton”or terminal and “post-synaptic density” are closely apposed (synaptic cleft only ~20 nm across) • Appropriate neurotransmitter receptors are clustered by different “scaffolding” molecules (not rapsyn; also MuSK is a muscle-specific kinase)

Question 6

What are the scaffolding proteins at CNS synapses?

Answer 6

• Glycine receptors are clustered by gephyrin
• Gephyrin is also present at GABAergic synapses but it is probably involved in clustering only a subset of GABA receptors
• PSD-95 at excitatory synapses: binds NMDA glutamate receptors directly and AMPA glutamate receptors indirectly

-NMDA receptors are physically bound to psd95

Question 7

What are the stages of spine synaptogenesis?

Answer 7

• 1. outgrowth of transient dendritic filopodia
• 2. stabilization of filopodium by contact with an appropriate axon, preventing retraction
• 3. formation of nascent synapses on filopodia and assembly of pre- and post-synaptic specializations
• 4. conversion of filopodia into mature spines by synaptic activity
• 5. activity-dependentrefinement

Question 8

How are initial contacts between axons and dendritic filopodia stabilized into synapses?

Answer 8

• Growing axons and rapidly extending dendritic filopodia appear to “search” the local environment • At contacts that will become stable, local calcium “flashes” observed when contact is made – recognition signals not known but neurotransmitter (e.g. glutamate) not secreted yet • No basal lamina - direct contact possible between surface adhesion molecules e.g. Cadherins and other “synaptogenic” binding partners e.g. Neurexins/neuroligins (e.g neuroligin 1 becomes enriched in spine heads – Title slide) • Pre-formed “transport packets” of active zone proteins and post-synaptic receptors and scaffolding proteins are rapidly transported to developing synapse:not every molecule individually -have pre fromed packets -in trafficking vesicles

Question 9

PIC4What are the synaptic contacts like?

Answer 9

-froming synapse: -adhesion molecuels liek casdherins that bind to themselevs= homofilic reaction, helps the synapstic sides stick together -or can have neurexin and neurolignin= heterophilic also conenct teh synpase togetehr

Question 10

What are the known regulators of synaptogenesis?

Answer 10

• this is the complexity= don’t have to know
• can have many combinations in what binds the synapse together
• these interactions thought to be early on in synaptic development

Question 11

How is the recognition, stabilization and functional development of CNS synapses complex?

Answer 11

• Many different cell surface proteins involved (homophilic – binds same type AND heterophilic – binds a different protein) • Specific sub-types of excitatory/inhibitory synapses likely to arise from different combinations e.g. specificity of particular neurexin splice forms (>2000 predicted) for GABA receptors clustered with gephyrin and neuroligin 2 • New proteins involved in synaptogenesis being discovered, tested in cultured cell systems e.g Seizure related gene 6 (Sez-6)

Question 12

What are the types of synapses and what are they defined by?

Answer 12

–excitatory and inhibitory are on different parts of the axons -Different neurotransmitters broadly classified by their effect on the post-synaptic neuron • Excitatory synapses • Neurotransmitter glutamate, synapses on dendritic spines • Inhibitory synapses • Neurotransmitters GABA or glycine, synapses on dendritic shafts and cell body

Question 13

What is the effect of Sez6 in mice?

Answer 13

• Sez6 is made in developing cortex
• Neurons from Sez-6 knockout mouse have smaller responses to a given stimulus due to fewer synapses being there
• Sez-6 knockout neurons have less spines indicating fewer excitatory synapses
• Less PSD-95 staining also indicates fewer excitatory synapses in Sez-6 KO cortex
• Secreted Sez-6 enhances synaptogenesis through α2δ-1 binding (enhances excitatory synapses)

Question 14

What is the TSP role in alpha2delta1?

Answer 14

• different synaptic patterns of binding
• this is just froma different review
• TSP by binding to alpha2delta-1 can increase developmental synpatogenesis

Question 15

What is the time course of synaptogenesis?

Answer 15

-many synapses formed rapidly in the early postnatal period, in mice in the forst 3 weeks, then peak, plateau, refinement and then some are taken out -‘• Main period of synaptogenesis is shortly after birth • Stabilization of synapses and refinement of synaptic connections into functional circuits is experience- dependent

Question 16

Does synapse elimination happen in CNS?

Answer 16

• NMJ: poly-innervation to mono-innervation through competition, ultimately one motor neuron per muscle cell • Synapse elimination (or, more accurately, synapse editing) is a feature of CNS, even though most cells innervated by multiple neurons

Question 17

What is the synapse elimination like?

Answer 17

• During development, net increase in synapse formation but also synapse loss • Activity-dependent refinement so that inputs and outputs become matched: – connections originally overproduced – performance improved by adjusting numbers of connections, boundaries and strength of connections – can also involve loss of neurons by apoptosis, axon withdrawal as well as synapse elimination -synapse elimination allows efficiency in the nervous system

Question 18

What is the synaptic refinement like in the visual system?

Answer 18

• Visual system continues to develop postnatally • Sensory input (activity) refines connections to mature form • Before eye-opening, less well-organised system

Question 19

What is the visual field like in a mature visual system?

Answer 19

• Each eyeball sees both left and right visual fields • Signals from left visual field transmitted via right optic tract and vice versa

Question 20

What are the mature visual pathways?

Answer 20

• Retinal ganglion cells (RGCs) are the output neurons of the retina • RGC axons project to thalamus (lateral geniculate nucleus, LGN) • LGN neurons project to visual cortex in occipital lobe

Question 21

What is the layering in LGN?

Answer 21

• Left visual field from each eye comes together in right lateral geniculate nucleus • Inputs from each eye separate into discrete layers • Layers 1, 4, 6: left eye • Layers 2, 3, 5: right eye

Question 22

How can you identify the nerve terminals in the visual system?

Answer 22

-this is how you can colour the cortex, can label the terminals • Inject radioactive amino acid (3H-proline) into one eye • 3H-proline transported to retinal ganglion terminals to show layering • Released from terminals, taken up by LGN neurons, shows pattern of LGN terminals in cortex

Question 23

What is the visual cortex like?

Answer 23

• LGN neurons project to visual cortex (layer IV) • Within layer IV, terminals from LGN neurons in pathway from the labelled eye form ‘zebra stripe’ pattern, interspersed with unlabelled regions (terminals from unlabelled pathway)

Question 24

What are the ocular dominance columns?

Answer 24

• In cross-section, “stripes” map ocular dominance columns • Within a stripe, layer IV neurons are synaptically driven by the labelled eye, within interspersed unlabelled regions, the other eye • Ocular dominance columns established postnatally

Question 25

How are ocular dominance columns established?

Answer 25

• Temporarily covering one eye in a newborn kitten causes blindness in that eye when tested six months later • Everything is normal in retina and LGN, blindness is cortical • Ocular dominance columns abnormal • Blindness is permanent

Question 26

What is the effect of temporary eye closure?

Answer 26

• Normal ocular dominance map shared 50/50 between eyes (labeled vs unlabelled) After temporary eye closure: • Deprived eye has greatly reduced ocular dominance map • Open eye has greatly enhanced ocular dominance map

Question 27

What is the effect of timing of eye closure?

Answer 27

• Close one eye at birth in kitten, open eye occupies 100% of cortical area • Close at 2, or 3 weeks has progressively weaker effect • Closure at 6 weeks has no effect • Defines critical period where left and right eyes sort out ocular dominance columns

Question 28

What is the critical period?

Answer 28

Critical period: a time window of enhanced plasticity during which experience (neuronal activity) rearranges developing circuits, permanently altering performance • Initial overlap is 100%, by end of critical period, terminals are segregated in layer IV (closing eye in adult has no effect) • Depends on relative activity, not absolute (competitive process) • If both eyes closed, spontaneous (non-light driven) activity of retinal ganglion cells can still cause rough columns to form but need visual input to get sharp borders

Question 29

What is binocular vision and how is it achieved?

Answer 29

need to integrate the information from both eyes -the neurons that are in layer IV have projections to more superficial layers -most neurons in layer III get input from both eyes= convergence of information -layer 3 is getting binocular input • In LGN and layer IV of cortex, left and right eyes kept separate • Vision is monocular to this stage • Cells in layer IV converge onto neurons in layer III • Most layer III neuron gets inputs from L IV ocular dominance columns from both eyes • Vision in layer III is binocular

Question 30

What is the mechanism of the Hebbian synapse?

Answer 30

* Donald Hebb proposed that competition depends on relative timing of activation of synapses * Originally proposed to explain learning and memory, but seems a more general phenomenon * “Neurons that fire together, wire together” but also need to consider that this is only true if the firing of neuron A helps neuron B fire - left eye= strong input, then B fires more - if right fires out of sync= then weaker connection - punishment and reward system

Question 31

What is the punishment and reward system?

Answer 31

• NMJ: both “punishment” and “reward” signals required in model for competition between multiply-innervating neurons • Evidence (Je HS et al., PNAS, Sept. 2012) suggests that proBDNF acting on p75NTR is punishment signal while mature (processed) BDNF is reward signal at NMJ • Mechanism may also be applicable to CNS synapse -neurotrophins are involved, neurotrophins can be the reward

Question 32

What is the neurotrophic reward model?

Answer 32

- neurotrophin in proform= can be punishment signal via p75 - mature form= reward via Trk receptors - weaker inputs get weaker by getting punished by the stronger inputs - Evidence from developing visual system: * Adding BDNF or NT4 to visual cortex prevents formation of ocular dominance columns (swamps normal small signal) * Blocking TrkB receptors also interferes

Question 33

What is strabismus and synaptic competition?

Answer 33

• In layer III,binocular cells sort out inputs on Hebbian principles • If L III neurons receive inputs from both eyes and these are firing in synchrony, both these input synapses are likely to be strengthened – binocular vision • If the eyes are misaligned e.g strabismus (a.k.a. cross-eyed or wall- eyed), synchronicity of firing is disrupted (as eyes looking at different targets) and binocular vision is lost -if get firing at the same time from both eyes= then get the binocular inputs is not created

Question 34

What is the effect of strabismus on layer II cells?

Answer 34

• After strabismus, no co-ordinated firing between inputs from each eye • Competition occurs between eyes in layer III • Winner takes all:most layer III neurons innervated by inputs from either one or the other eye (monocular inputs)

Question 35

What are the critical periods in human development?

Answer 35

• Cataracts or strabismus in neonates lead to permanent visual problems if not corrected in first six months (peak of development of binocular vision ~3-4 months of age, if corrected between 1-2 years, get incomplete recovery) • Other sensory modalities have critical periods, e.g, hearing, language

Question 36

Summary I?

Answer 36

• CNS neurons receive many hundreds of synapses (excitatory – on dendritic spines, inhibitory – on shaft or neuron cell body) • Dynamic filopodia are stabilized by contact with appropriate axons to form spine synapses • Developing synapses can be assembled rapidly but functional maturation takes days to weeks and is experience dependent

Question 37

Summary II?

Answer 37

• Changes in synaptic efficacy and synaptic elimination are important in development (and in the plasticity of the adult brain) • Both result from competition between synapses (punishment and reward likely to involve proneurotrophins and neurotrophins, respectively) • Development of ocular dominance columns in L IV of visual cortex and binocular inputs in L III are permanently compromised if sensory input (vision) is impaired during early post-natal period (critical period) when plasticity is highest