SMITH Stephen Connectome 28 Nov 2011

title: the synaptome meets the connectomes

jay... i thot there was only one connectome!

one based on dti...

plural connectomes  cuz it depends on the technique... microtracing, dti, virus tracing etc.

topic one... synapse complexity and diversity...  story has chnaged in past two decades...

sl 1 (slide 1)...  in the 1950s...   saw synapse for the first time in EM...
historic foto from thomas reese...

PREsynaptic bouton with vesicles and a presynaptic active zone... voltage dependent calc. channels cause fusion...

sl:  synaptic vescle is studded with scores of proteins in this cartoon... a microcosm on its own... (that figure is online... see images)

presynaptic active zone...  also incred complex network involving exocytosis  and endocytosis...

even the synaptic cleft is quite complex... with various adhesion molecules

cool picture of synap vesicle is from 2006...on google.

also  postsynaptic density has vast complexity...

so, to summarize... hundreds (or maybe even thousands) of protein species in each synapse.

and in any one synapse... u may have 10**5 proteins among perhaps hundreds of protein categories.

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kinases, phosphatases, phospholipids... many have memories...

synapse is daring u to oversimplify it.

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synaptic molecules r diverse...  1920 to 1980.. discovered difft neurotransmitters. (glutamate was among the last)

glutamate at 90%
gaba at 10%

1980 to present...

protein and isoform diversity... ion channels, ca buffers, vesicle proteins,

each synapse is like an entire microprocessor....

he uses 500 trillion has number of synapses...  (somewhere btwn 10**14 and 10**15)

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multiply 100 to 1000 Trillion synapses * 10**5  proteins per synapse...
he calls each protein a switch  =  total # of computers on earth...

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Ravi Iyengar...  circuit diagram of synapse...
signaling network in one synapse  hundreds of nodes and arcs...

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1970 to present...
dynamics and modulation...

release probability, current kinetics, voltage-dependence, facilitation, depression,
modulation, spine dynamics...

(1920 to 1970  was just excitatory vs inhibitory)

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So, what corners can we cut?  unknown  (in trying to understand function)

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he says "what u think of as individual syanpses are not"
neuromusc junction, squid giant axon, held junctions... et al  are all parallel arrays, sometimes of hundreds of synapses.

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TOPIC TWO:

ARRAY TOMOGRAPHY... invented at Stanford... his favorite tool...

embed brain in plastic (eg mouse brain) ... allows antibodies to get in and recognize targets...

thin sections float on water...  30 nanometers thick... a few dozen atoms thick... allow electrons to fly thru;

get long ribbons of serial sections

put on glass cover slip and then cover with immuno stains  + fluorescence.

say 4 difft colors... moving all nite long...

stitch and align 3d mosaic... finally

get  multi-color volume image stacks...

similar to confocal scope BUT

min voxel is about 2% of size of confocal  and no depth dependence...

30 to 70 nano is the thickness of the slice  (compares to 700 nanos or worse with confocal)

lateral resolution is  better by a factor two or more than confocal  (cuz no spherical aberration);

dimensions are big compared to proteins but small compared to synapse...

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can ERASE the first batch of immuno antibodies labels  (dissolve them off by pH change) then apply the NEXT four...
can get up to perhaps 60 difft antibodies of same slices....

he compares two images of anti-synapsin 1 immunostained mouse cortex neuropil...

looking at a box abt 25 microns on a side...  array is far better than confocal...

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great series of clips...  mouse brain....

he shows gumstick image...  it is 1.5 mm long from pia down to striatum...

gum stick contains abt 6M synapses...

human is 4000 X  volume of mouse brain...

gumstick is 1/50,000 of mouse brain...

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thy-1 YFP  ....  stained with green fluor protein (from birth)

only abt 3% of neurons express this protein...

even this slide  only shows abt 3% of synapses...

slide shows a crop of pyramidal cells

main cell that expresses this are layer 5b pyram neurons...

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anti-tubulin...  stains tubules... used for transport... (dfusion is too slow... this is a rail road system)

this shows axon or dendrite of 100% of neurons
shows in blue

then in red...
anti=synapsin1... sea of red... voids are neural bodies...

one billion in every microliter  (synapses)

40 dimensional space  (40 markers)  a synaptogram....

neural cell bodies are festooned with inhibitory synapses  (gabaergic)

cholinergic  and dopamnergic are rare but pack a wallop...

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psd95  is a marker of post synaptic density...  and also stain with synapsin... to determine the axis of the synapse.

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good reason to think that synapses r what store memories...
but don't know exact mechanism...

dynamics from snapshots... he shows tall towers with construction cranes..

there are many plasticity marker candidates...

he shows pic of dtr... catherine rose smith

back to the gumstick...

lovely 4 minute youtube...

red synapsin. labels the synapses.