Post on 12-Mar-2020
Nanoscale analysis of
structural synaptic plasticity
Kristen M. Harris
Department of Neuroscience
University of Texas at Austin
synapseweb.clm.utexas.edu
NSE 2016
Hippocampal CA1 Pyramidal Cell
Neuron
Cell Body
Dendrites
DendritesDendritic
Spine
Axonal
Bouton
Adult Rat
Rapid Golgi preparation (Harris, 1980)
Axons
Dendritic Spines in Hippocampal CA1 Neuropil
1 micron
PSD
Glia
Axon
Spine
Dendrite
Glia
Adult Rat
Dendritic Spines in Hippocampal CA1 Neuropil
1 micron
PSD
Glia
Axon
Dendrite
Glia
Adult Rat
Dendritic Spines in Hippocampal Neuropil
SER
Spine
Apparatus
dcv
0.5 m
Do synapse size and composition
reflect function?
John Fiala
http://synapseweb.clm.utexas.edu > Tools > Software
Reconstruct
Introductory Cruisethrough
Hippocampal CA1
neuropil
Credits
University of Texas at Austin
Josef Spacek, Kristen Harris, Larry Lindsey, Patrick Parker
Chandra Bajaj, Jarred Bowden
The Salk Institute
Justin Kinney*, Tom Bartol, Terry Sejnowski
Dan Keller, Varum Chaturvedi
3D – Blender
Music by: Camille Saint Saëns, Carnival of the Animals, Aquarium
Josef Spacek
Charles University
Hradec Kralovè
Czech Republic
CA1 densely reconstructed volume.
Perfused 1984(~180 µm3)
Harris et al. (2015) (Nature) Sci. Data
~500 (498) synapses in volume = 1 RBC!!
(~180 µm3) Perfused 1984
Harris et al. (2015) (Nature) Sci. Data
Learning from 3DEM and realistic
MCell models.
Tom Bartol, Cailey Bromer,
Terry Sejnowski, Salk Institute
Mary Kennedy, CalTech
Bartol et al. (2015) Nanoconnectomic upper bound... eLife: 10.7554.
Bartol et al. (2015) Computational reconstitution of spine calcium... Front. Syn. Neu.:10.3389
Nature’s experiment provides coupled
spines with “same” activation histories.
Bartol et al. 2015b, eLIFE
Axon-coupled spines have nearly
equal head and synapse sizes.
Enhanced Cell Blender Axon-coupled spines
Fig. 3: Coupled spines
have nearly equal head
sizes. Variation across
population gives ~4.7
storage bits per spine.
Bartol et al. 2015, eLIFE
CV (0.083)
Uncoupled spines show no correlations.
Non-axon-coupled
different dendritesNot different from random
Bartol et al. 2015, eLIFE
26 distinct intervals in synapse size =
4.7 information bits available to CA1 synapses.
• CV (0.083) per distribution matches axon-coupled same dendrite spines.
• Intervals spaced to achieve 31% overlap with adjacent intervals.
• Gives 69% between peaks (discrimination threshold of information theory).
60 – fold range in size
Bartol et al. 2015b, eLIFE
MCell reconstitution models reveal fast Ca+2
transients missed by Ca+2 indicator dyes.
Small Spine 6x greater
[Ca2+] thanlarge spine
Bartol et al. 2015, Front. Synaptic Neuroscience
Conclusion 1
• Synapse size is a reasonable predictor of strength.
• Synapses are not simple on/off switches, but graded responders.
www.openconnectomeproject.org/synapseweb
Harris et al. (2015)
Scientific Data doi:
10.1038/sdata.2015.46
Control and Test pulses
at 1 pulse per 2 min
From looking and modeling to experiments.
Theta-Burst Stimulation
(TBS) – 8 trains
S1
S2
0
50
100
150
200
250
300
350
-40 -20 0 20 40 60 80 100 120
% B
as
elin
e
Time (min)
Time series 3DEM of LTP
ControlLTP
Bell et al. (2014)
Bourne and Harris (2011)
Jensen and Harris (1989)
Microwave-enhanced fix at key times.
5 min 30 min 2 hr
Chair, Dept. Neurology
Perelman Sch. Med. Univ. Penn.
Frances Jensen
Jen Bourne
Univ. Colo.
Denver
TBS
Beth Bell
EA, Austin
~120 µm from LTP or Control Stimulating Electrodes
Investigate regions with
differential activation.500 µm
70 µm
CODED:
HCSBRUnbiased
length
Dendrites with TBS-LTP had fewer
spines with larger PSDs than controls.
Control TBS-LTP
50th
percentile
dendrites
At 2 hours,
but not at
5 or 30 min.
Conclusion 2
Many small
synapses
Fewer large
synapses
Adult dendrites have limited total synaptic input.
Control TBS-LTP
Equal total PSD area across
time and conditions.
50
100
150
200
250
-40 -20 0 20 40 60 80 100 120
% B
as
eli
ne
Time (min)
What structural changes underlie
‘silent’ growth of the PSD?
Control
LTP
TBS
Synapse strength
does not increase
beyond the first 5
minutes after TBS.
Typical view of a spine synapse with
thickened postsynaptic density (PSD).
50-60 nm
Section
Thickness
250 nm
AZ = PSD across from the active zone with vesicles.
Serial sections through that PSD.
AZ = PSD across from the active zone with vesicles.
250 nm
Nascent Zone (NZ) revealed in serial
sections through the PSD.
250 nm
NZ = PSD across from no presynaptic vesicles.
Nascent Zone (NZ) revealed in serial
sections through the PSD.
250 nm
NZ = PSD across from no presynaptic vesicles.
Nascent Zone (NZ) revealed in serial
sections through the PSD.
250 nm
NZ = PSD across from no presynaptic vesicles.
Role of Nascent zones in
“silent” PSD growth.
Bell, et al., (2014)
Spacek and Harris, (1998)
Beth Bell
Josef Spacek
Arial ViewLateral View
NZ AZ
Silent synapse growth by addition of
nascent zones?
Stochastic M-Cell modeling: Probability of AMPA-type channel opening falls
off exponentially from 0.4 at the center of release to 0.1 at 200 nm away.
(Franks et al., 2002, 03)
Small dense core vesicles (DCV) carry
presynaptic active zone proteins.
DCVs transport AZ proteins
(Piccolo, Bassoon)DCV spicules
Sorra, Mishra, Kirov, Harris (2006)
Karin Sorra
Arroscience
DCV in Transport Packet
1 µm
(C. Garner, N. Ziv and Others)
Surprisingly, DCVs occur throughout
mature CA1 axons.
Sorra, Mishra, Kirov, Harris (2006)
1 µm3
DCVs are recruited to presynpatic
boutons by 5 minutes after TBS.
Return to control levels
by 30 min.
Sometimes, DCVs ‘inserting’
near edge of the PSD.
Bell et al., (2014)
1 µm
Addition of presynaptic vesicles converts
NZs to AZs, with no change in PSD area.
NZ: LTP < ControlPSD area:
LTP = Control
Control TBS-LTP
Growth of nascent zones is primary source
of postsynaptic growth in PSD area.
PSD: LTP > ControlNZ: LTP > Control
50
100
150
200
250
-40 -20 0 20 40 60 80 100 120
% B
as
eli
ne
Time (min)
Purpose of the silent
synapse growth following LTP?
Control
LTP
TBS
Synapse strength
does not increase
beyond the first 5
minutes after TBS.
LTP Saturation lasts >1 HR (LE Hoods)
No Augmentation - 30 Minutes No Augmentation - 1 hr
Cao and Harris, J. Neurophys. 2014
Adult Adult
Reliable augmentation of LTP
by 4 hours.
Third TBS at 4 hours
produces augmentation.
Augmentation begins at
90 minutes
Cao and Harris, 2014
Conclusion 3: Structural mechanism for
advantage of spaced vs massed learning.
Augment LTPSaturates LTP8 TBS
by 5 min by 30 min by 2 hours
NZ conversion to AZ underlies LTP.
Silent NZ growth prepares synapse for
enhanced LTP augmentation and learning.
Improving Electron Microscopy:
Scanning Electron microscopy in
the Transmission mode (tSEM)
Kuwajima , Mendenhall , Lindsey Harris (2013) PLOS 1
John MendenhallMasa Kuwajima
Neuropil in tSEM at
nanometer scale
Kuwajima M, Mendenhall JM, Lindsey LF, Harris KM (2013)Randy Chitwood, UT-Austin
Standard
TEM field
tSEM
field
Kuwajima , Mendenhall , Lindsey Harris (2013)
Rationale for expanding tSEM.
• Large fields at high resolution with little or no
montaging.
• Low dosage kV and small beam size produce
little or no section shrinkage during imaging.
• Automated image collection across serial
sections at <2 nm x-y resolution.
• Transmission retains all information
throughout the section thickness.
Bottlenecks and improvement.
• Fragile thin sections.
• Disambiguation of obliquely sectioned objects
having borders within thin sections.
• Alignment of large tSEM fields.
Thicker sections are less fragile but
have more ambiguous membranes.
150 nm
ves?
ecs?
sa?
mt?
Tomography reveals buried structures in
15 nm virtual sections.
Thicker sections plus tomography solution.
sasa
Automated large field alignments
being solved!
Wetzel, Bartol, Sejnowski
Large field alignment, yellow box
– prior size limit.
Conclusion 4
• The future is bright for experimental 3DEM!
0.5 m
Current Lab:
Lyndsey Kirk
Masa Kuwajima
John Mendenhall
Patrick Parker
Clayton Smith
Seth Weisberg
Corey Haines
Paola Gonzolez
Marshall Drake
Dakota Hankama
Sindy Ventura
Dusten Hubbard
Kate Dempsey
Alyssa Norbert
Thanks!
Sergei Kirov
Yelena Kulik
Anusha Mishra
Linnaea Ostroff
Lara Petrak
Chris Risher
David Selig
Gordon Shepherd
Bitao Shi
Heather Smith
Karin Sorra
Beatrice Tsao
Rachel Ventura
Mikayla Waters
Deborah Watson
Mark Witcher
NS21184, MH095980, NS074644,
Kavli and Brain Research Foundations
Texas Emerging Technologies Fund
Collaborators:
Josef Spacek
Cliff Abraham
Terry Sejnowski
Tom Bartol
Mary Kennedy
Cailey Bromer
Chandrajit Bajaj
Randy Chitwood
Dan Johnston
Boris Zemelman
Joshua Vogelstein
Randal Burns
Alex Baden
Former:
Jennifer Bourne
Beth Bell
Jared Bowden
Albert Cardona
Guan Cao
Marina Chicurel
Michael Chirillo
Mitya Chklovskii
James Cooney
Mike Ehlers
John Fiala
Alex Goddard
Jamie Hurlburt
Frances Jensen
Paul Jackson
Larry Lindsey
Olga Ostrovsky (3/17)
Max Snodderly
Guan’s Goodbye Party