Spatial constraints dictate glial territories at murine - PowerPoint PPT Presentation

Spatial constraints dictate glial territories at murine neuromuscular junctions Brill MS, Lichtman JW, Thompson W, Zuo Y, Misgeld T.  Sebastian Anastassiou

Introduction  Glial cells have many functions around the nervous system.  Synaptic functions: Monitor neurotransmission  Contain and clear released transmitters  Modulate synaptogenesis and plasticity   Need a highly organized arrangement.  Many neurological diseases associated with altered glial morphology and arrangement, e.g. ALS, HD.  How glial cells establish and maintain their perisynaptic territories is not known.

NMJ ideal for study  NMJ ideal to study this due to accessibility and size.  Axonal and terminal Schwann cells (A/TSCs).  During development, SC are dynamic and proliferate.  During adulthood, SC numbers are stable.  Denervation causes reactive transformation of SC, proliferation and growth of processes.  Essential for guiding regenerating axons (bridge).

Aims  What is the territory of individual terminal SC under normal conditions.  How is this territory established during development, and what mechanisms maintain it.  How single SC territories change after axonal degeneration, and which signals drive these changes.

Methods Transgenic Mice  Transgenic SC-GFP mice, express GFP in SCs.  Crossbred with thy1 -XFP mice to label axons.  Transgenic Δ NLS mice – variation of Wlds protein to delay axon fragmentation after axotomy.

Methods Viewing individual SC territories  Sequential photobleaching of SCs.  Confocal microscope  Laser  Sequential dye-filling of SCs.  Rhodamine dextran

Methods In-vivo NMJ imaging  Time-lapse confocal microscopy.  Combined with photobleaching. SC ablation  Laser pulse

Results 

Experiment 1 and 2  Used sequential photobleaching and dye filling methods to image SCs.  Aim: to compare morphologies of immature and mature TSCs.  Hypothesis 1: TSCs arrange themselves in a highly organized tile- like manner.  Hypothesis 2: Two processes by which this arrangement could emerge: SC territories are already segregated at immature NMJ as SCs  emerge sequentially by local proliferation. SC territories are initially intermingled and then segregate as  development progresses.

Experiment 3  These morphological differences led to hypothesis that cell dynamics were either involved in or even responsible for the remodeling.  Used time-lapse imaging of the triangularis sterni muscles.

Experiment 4  What determines SC partitioning of the NMJ during these different developmental stages?  Hypothesis: Competition for perisynaptic space during SC segregation determines SC partitioning of NMJ.  Used the SC ablation method to destroy single SCs.

 Suggests mature terminal SCs’ lack of dynamism is due to spatial competition.  Axonal SCs are restricted by additional factors at the heminode or by intrinsic factors as a result of their differentiation.

Experiment 5  SCs could also be spatially constrained by axons.  Hypothesis: removing axons would result in SC dynamism and intermingling.  Axotomized motor neurons.

 Less dramatic than SC ablation, but still showed fast volume expansion of SCs.  Suggests also underlying axon prevents SC intermingling.  SCs explore vacated gutter first then surrounding area following axon removal.

Experiment 6  Axotomy leads to vacation of synaptic space as well as loss of neural activity.  Axons maintain SC segregation via neural activity or axon presence?  Blocked NT with BoTX A.  Used the Δ NLS mice.

 SC segregation is independent of neuronal activity.

Conclusions  Mature SCs are static and arranged in a tiled manner around synapses.  Immature SCs are dynamic, exploring synaptic and extrasynaptic territory.  The mature SC arrangement is maintained by competition for perisynaptic space through axon-glial and glial-glial interactions.

Constraining SCs  Basal lamina laterally constrains SCs.  Heminode prevents retrograde growth by TSCs.  None of these likely to constrain individual TSCs.  Space filling model rather than homotypic repulsion model: No continuous expanding and retracting – TSCs in permanent  contact. Axon removal induces SC expansion.  Immature TSCs intermingle. 

Big Burning Question  What regulates the neonatal dynamism and adult plasticity of terminal Schwann cells?

Exploratory behaviour of immature TSCs  Immature axon terminals very dynamic:  Synapse elimination frees up perisynaptic space.  Segregation of terminals from different motor axons may need some compensatory glial dynamism.  Seems to be a looser synaptic cell arrangement as axon terminals extend and retract small processes readily.  Gradual development of the basal lamina allows immature axonal and glial processes to be sent outside synaptic boundaries.

Mature TSCs  Spatial competition prevents mature TSC expansion.  Cannot exclude glial expansion induced by factors released following cell ablation.  Unpredicted discovery of phagocytic capacity.

TSCs as models for glial function  Results show similarities between TSCs and CNS microglia and astrocytes:  Phagocytic capacity.  Static and dynamic states at immature and mature stages respectively.  Non-overlapping space filling arrangement.

Shortcomings  Obtaining cell outlines by image subtraction is prone to noise, creating uncertainty in fine detail.  Possible phototoxicity by photobleaching (although evidence suggests it does not occur).

Future Studies  Use TSCs as disease models, e.g. ALS, HD.  Investigate TSC proliferation and expansion with more chronic cell ablations.  Investigate how this affects synaptic function.  Investigate role of axon in SC segregation more since there seems to be a slight delay before cell expansion occurs.

Reversing
the
outcome
of
synap4c Elimina4on
at
the
developing neuromuscular
junc4ons
in
vivo: evidence
for
synap4c
compe44on and
its
mechanism Stephen
G.
Turney
and
Jeff
W.
Lichtman Presented
by:
Brodie
Ballan4ne

Background • Prior
to
this
paper
lots
of
evidence
that
the events
that
cause
mul4ple
synap4c
connec4ons to
just
a
single
connec4on
at
the
NMJ
is determined
by
local
mechanisms
at
each individual
NMJ. • A
few
hypotheses
of
what
this
mechanisms
are: 1.Random
scale
back
of
arbors 2.Fate
predetermined
by
posi4onal
or
molecular cues 3.Compe44on

Aims • Test
if
synap4c
elimina4on
is
driven
by
interaxonal compe44on • When
does
this
elimina4on
process
finally
become irreversible? Method
–
abla4ng
axon
with
the
higher
chance
of
being maintained
and
seeing
if
fate
of
other
is
reversed.

Laser • Diode‐pumped
mode
locked
Ti:sapphire
laser
oscillator • Focused
laser
over
axon
using
scanning
microscope
system • Axons
fluorescence
was
bleached,
causing
damage
to
one axon
leaving
adjacent
undamaged. • 30‐45
minutes • Caused
die
back
similar
to
that
seen
in
acute
axon degenera4on.

Experiment
1 • Using
mice
7‐8days
old • They
located
NMJs
that
were
innervated
my
two
axons • 87
experiments
failed • 15
succeeded 10/15
larger
caliber
was
ablated
(easy
to
see
which
was had
larger
caliber)6
of
which
accompanied
less
than 5% 4/15
around
the
same
caliber
(based
of
appearance
and other
factors
they
could
Iden4fy
the
one
that
had
less territory 1/15
ablated
the
axon
with
the
small
caliber

Experiment
1
cont. • How
long
did
remaining
axon
con4nue
to
lose territory
for
un4l
changing
its
fate? • They
reimaged
3
junc4ons
less
than
24
hours. • One
at
6,
one
at
12
and
one
at
17h • Even
at
6
hours
the
remaining
axon
had
lost no
territory

Results • In
12/13
axons
images
aeer
24
hours
the remaining
axon
accompanied
the
en4re
synap4c site. • The
other
accompanied
75% • Along
with
thickening
of
the
caliber
of
its preterminal
branch Conclusion Once
one
of
the
compe4ng
axons
(dominant
or not)
are
removed
the
remaining
changes
its
fate to
become
the
dominant
axon
with
no
lag
4me.

Experiment
2 • Abla4ng
of
singly
innervated
NMJ
while
other axon
recently
eliminated
and
retrac4ng • Are
the
retrac4ng
axons
irreversible
or
will
they reverse
there
fate
again? • Of
the
18
examples
55%
of
the
retrac4ng
axons reversed
their
fates,
grew
back
and
reinnervated the
whole
junc4on • It
was
shown
however
that
the
further
away from
the
junc4on
the
retrac4ng
axon
was
the less
chance
it
would
reinnervate.