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)
- MORPHOLOGY OF NEWLY FORMED SYNAPSES - what are the main features of a mature synapse and how these develop?
- PROGRESSION OF SYNAPTOGENESIS - where are pre and post synaptic contacts initially formed and how does synaptogenesis progress?
- EARLY SIGNS OF SYNAPTIC FUNCTION - how is neurotransmitter release triggered and how early does it occur following a contact?
- 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)
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
- growth cone contacts myotube
- neural agrin induces ACh receptors to cluster
- 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:
- nt is packaged inside small vesicles in the presynaptic nerve terminal
- when an action potential invades the nerve terminal, voltage gated calcium channels open and calcium flows into the cell
- 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