synaptogenesis, synapse function and refinement

BASICS

reticuar vs neuron theories

synapse formation begins with axonal and dendritic growth cones following a set of extracellular cues to target a specific region

when the axon growth cone contacts an appropriate postsynaptic cell, a decision is made to stop growing and to differentiate into a presynaptic terminal

neurons don't act syncitially

a separation between nerve cells allows for intercellular communication (chemical synapse)

complexity of neuronal connections in the brain

majority of synapses are chemical - neurotransmitters are secreted by the presynaptic neuron and detected by the postsynaptic neuron

excitatory and inhibitory synapses

GABA = main inhibitory neurotransmitter

glutamate receiving are peripheral, inhibitory are close to soma

glutamate = excitatory neurotransmitter

proximal part of axon (AIS - where axon potentials are initiated) - lots of inhibitory synapses so that over excitation doesn't occur

most excitatory synapses are on spines

techniques that help to identify and study synaptic connection

electron microsope (see the complex structures of synaptic contacts at ultrastructural level)

live imaging (monitor synaptogenesis over time)

intracellular recordings (observe their electrical behaviour)

  1. MORPHOLOGY OF NEWLY FORMED SYNAPSES - what are the main features of a mature synapse and how these develop?
  1. PROGRESSION OF SYNAPTOGENESIS - where are pre and post synaptic contacts initially formed and how does synaptogenesis progress?
  1. EARLY SIGNS OF SYNAPTIC FUNCTION - how is neurotransmitter release triggered and how early does it occur following a contact?
  1. REFINEMENT OF CONNECTIONS

why is synaptic refinement important and what are the driving forces that shape synaptic connections

signs of synaptic specialisation at ultrastructural level

accumulation of small vesicles at the presynaptic site

formation of a narrow cleft filled with extracellular matrix between pre and post synaptic membranes

thickening of the postsynaptic membrane owing to the accumulation of membrane associated proteins and cytoskeletal elements - postsynaptic density

different synapses develop at different timescales

development of synapse morphology

newly formed synapses do not display any of the adult anatomical features of a synapse

newly formed synapses have few, if any, vesicles in the presynaptic terminal

there is a close apposition of pre and post synaptic membranes (a tight junction)

postsynaptic membrane does not yet display a postsynaptic density

takes days -> weeks

example contact of a mouse auditory nerve end bulb onto its postsynaptic target (spherical bushy cell) image

stages of synapse formation

the structure of a synapse appears to mature over a relatively long period (initially presents few features of a mature synapse)

first signs of differentiation appear on the postsynaptic membrane

accumulation of Golgi apparatus

appearance of coated vesicles

postsynaptic cell is FIRST to display any signs of differentiation

membrane and surface glycoproteins - synaptic inductive factors, promoting pre-post synaptic differentiation

receptors must arrive in postsynaptic membrane through vesicles - made of glycoproteins

types of synapses within the central nervous system

common

axodendritic

axosomatic

less common

axo-axonic

dendro-dendritic

axon initial segment (AIS) contact are GABA releasing to inhibit over excitation

most rare

most common

where are contacts initially formed

in many systems including the neuromuscular junction, spinal cord, hippocampus and cortex, contacts seem to form initially on postsynaptic filopodia

excitatory glutamatergic synapses are found on dendritic spines, whereas inhibitory GABAergic synapses tend to localise near the cell body and on dendritic shaft

creates the question - where are contacts first formed?

filopodia

small protrusions from soma and dendrite

exploratory, very dynamic

first contact

myopodia

filopodia on muscle fibres

first contact between motor neuron and five

eventually reaches full synaptic junction

at the end of filopodia psd95 expression shows contact has been made (GFP imaging)

how does synaptogenesis progress?

a process of trial and error

branches are stabilised when synapses mature on them

mature synapses are the sites of new branch addition

the synaptotropic hypothesis proposes that input from a presynaptic to a postsynaptic cell eventually can change the course of synapse formation at dendritic and axonal arbours

synapse formation is required for the development of neuronal structure in the functioning brain

some contacts are stabilised, innappropriate contacts will be removed

removing the synapse automatically removes the filopodium

stabilised filopodium generates new filopodia looking for possible partners

if stabilised new filopodia generate from there

synapse formation and neuronal morphology

depends on the synaptic connections a cell is able to make with presynaptic neurons

when does pre and post-synaptic differentiation occur?

one advantage of in vitro systems is that is is feasible to visualise contact events between pre and post synaptic neurons

imaging contact events in vivo is considerably challenging, particularly in the CNS

neuromuscular junction is more accessible to labelling and imaging

found that motor neurons, although they innervate clusters, pretty much always post synaptic differentiation appears first pre innervation

always exceptions - cases where we see post synaptic structures in the absence of presynaptic neurons

observations suggest the postsynaptic differentiation occurs in the absence of a presynaptic partner

most of the time we know post synaptic differentiates first

post synaptic differentiation requires the growth cone

important for clustering of receptors

motor neuron derived Agrin induces AChR aggregation

works in concert with Neuregulin, known to activate transcription of synaptic receptos

we know that Agrin is secreted by the presynaptic neuron, binding to MuSK receptors and dimerising them with LRP4 - activating rapsyn

rapsyn links multiple acetylecoholine receptors together

you need agrin secreted by the motor neuron or mature and organise the post synaptic receptor

  1. growth cone contacts myotube
  1. neural agrin induces ACh receptors to cluster
  1. synaptic basal lamina forms

the motor neuron specific form of agrin (cognate ligand for musk, proteoglycan) binds to two molecules of MuSK (receptor Tyrosine kinase), dimerising them along with molecule soft coreceptor LRP4

assembly of a MuSK/LRP4 complex autophosphorylates internal portion of MuSK which activates raspyn

raspin directly binds and anchors the cytoplasmic portion of several AChRs together and to the synaptic site

neurotransmitter release from presynaptic terminals

central component of neuronal communication:

  1. nt is packaged inside small vesicles in the presynaptic nerve terminal
  1. when an action potential invades the nerve terminal, voltage gated calcium channels open and calcium flows into the cell
  1. the influx of calcium triggers the fusion of the neurotransmitter-filled synaptic vesicles with the cell membrane, thereby releasing their contents into the synaptic cleft

neurotransmitter & recepto

glutamate -> NMDA, AMPA receptors

GABA -> GABA receptors

Ach -> AChRs

it is nearly impossible to record from a cell at the exact moment it is first contacted by the growth cone in vivo

all growth cones release a neurotransmitter

released spontaneously

patch clamp - technique measures membrane potential and amount of current passing across the cell membrane

spontaneous release of motor neuron neurotransmitters before the motor neuron has made contact with the post synaptic terminal - spontaneous post synaptic current is observed when two neurons are in close contact

muscle cell contact enhances spontaneous and evoked transmission

both the growth cone and postsynaptic cell generate many of the components needed for neurotransmission well before innervation occurs

stimulating motor neuron - immediately after contact see a small amplitude of PSC (post synaptic current), few minutes later muck bigger

spontaneous AND evoked PSC

both growth cone and postsynaptic cell generate many necessary components for communication pre innervation

why is refinement essential?

ensures that

each target cell is innervated by the 'right' number of axons

each axon innervates the 'right' number of target cells

refinement is the elimination of immature contacts

much on what we know about synaptic refinement came from simpler and more accessible parts of the nervous system eg. neuromuscular junction and peripheral nervous system

the process of refinement is not a net loss of synapses

removal of immature contacts from all but one or few axons on each target and the focus on fewer target cells by a progressively increasing amount of synaptic machinery for each axon that remains

1:1 relationship for motor neurons and muscle fibres - innervation might occur at mutiple targets but by maturation only one remains

refinement can also be seen in the CNS

eg. innervation of purkinje cells by single climbing fibre axon

multiple climbing fibres innervate form the inferior olivary nucleus synapse on purkinje cell somas

lots of cells pre pruning but ends up with only one

ALL POST NATAL

PRUNING OF SYNAPTIC CONNECTIONS IN THE HUMAN BRIAN

when we are born have a similar number of neurons to adulthood

during early childhood lots of synaptic contacts are made in the brain

period of synaptic pruning occurs between 4-6 years - infantile amnesia?

synaptic pruning is very important - specific forces drive it

trophic support and electrical activity sculpt connections

axons compete for a limited amount of trophic factors, such as neurotrophina, provided by the target cell

only contacts receiving sufficient amount of trophic support will survive and mature, rest is eliminated

fine tuning of synaptic connections is determined by the pattern of electrical activity in the presynaptic neurons. a post synaptic cell may initially be innervated by many different presynaptic cells

only presynaptic inputs whose activity is correlated in time with postsynaptic activity are maintiaed

three presynaptic neurons make contact

amount of neuronal activity differs

two have similar amounts of activity

one is asynchronous/diffeent amount of activity

lack of synchrony causes axonal input to eventually be eliminated

lots of other factors but key driver is electrical activity

immune signalling

MHC class I molecules and MHC receptor PirB

neuronal pentraxins - cyclic multimeric proteins involved in calcium dependent ligand binding

could be working together via microglia - resident macrophages of the CNS

engulf synaptic components from less active neurons

might clean up synaptic debris or play a more active role in pruning

by P11 in mice many somatic climbing fibre synapses are eliminates

a winning climbing fibre translocates and synapses on the purkinje cell dendrites

in second stage p12-p17 any remaining somatic climbing fiber synapses are pruned away (requires hebbian type plasticity)

strengthening of single climbing fibre input occurs, while other inputs weaken and are eliminated

climbing fibre input most synchronous to the purkinje neuron burst output becomes the 'winner'

increased GABAergic innervation onto the purkinje cell soma from cerebellar basket cells drives the relative weakening of the losing climbing fibre inputs during the first stage of pruning

during the later stage of pruning, signalling downstream of metabotropic glutamatergic receptors (mGluR1) in purkinje cell dendrites drives the pruning of any remaining somatic climbing finer synapses

mGluR1 singaling simulates expression of membrane tethered semaphore Sema7a and the release of Brain-derived neurotropic factor from purkinje neurons onto remaining somatic climbing fibre synapses to facilitate their removal

molecules eliminate losing climbing fibres and stabilise winning ones

Sema3A is seceted from purkinje neurons to promote stabilisation and maturation of winning climbing fibres throughout pruning stages

progranulin (derived from purkinje cells) binds to climbing fibres via Sort1 to strengthen and stabilise them

influence of glia particularly specialised cerebellar astrocytes called Bergman glia is crucial in strengthening the winning synapses and preventing excessive climbing finer innervation along purkinje cell dendrites during later pruning stages

additional influences of microglia promote the weakening of a subset of climbing fibre synapses

differential neuronal activity drives a competitive pruning process (demonstrated by retinogenicular and cerebellar climbing fibre circuits)

process of cerebellar climbing fibre pruning provides further insight into the activity dependent molecules that drive this competitive process