Neurotrophin-induced plasticity in the developing visual system:

from axons and dendrites to synapses

NeurotrophinNeurotrophin--induced plasticity in the induced plasticity in the developing visual system: developing visual system:

from axons and dendrites to synapsesfrom axons and dendrites to synapses

Susana CohenSusana Cohen--Cory, Ph.D.Cory, Ph.D.Dept. of Neurobiology and BehaviorDept. of Neurobiology and BehaviorUniversity of California, IrvineUniversity of California, Irvine

1. Targeting2. Branching3. Synaptogenesis

1 2 3

NeurotrophinsNeurotrophins and their receptorsand their receptors

Neurotrophins can induce multiple actions and activate multiple signaling mechanisms

Neuronal survivalNeuronal survivalDifferentiationDifferentiationAxonal/Axonal/dendriticdendritic growthgrowthSynaptic Synaptic potentiationpotentiation/plasticity/plasticity

BDNF signalingBDNF signaling((presynapticpresynaptic neuron)neuron)

↓↓ Synapse maturation & functionSynapse maturation & function

↓↓ Synapse stabilizationSynapse stabilization

↓↓ Arbor maturationArbor maturation

BDNF and the control of synaptic connectivityBDNF and the control of synaptic connectivity

↑↑ Synapse Synapse innervationinnervation densitydensity

↑↑ No. of postsynaptic partnersNo. of postsynaptic partners

↑↑ Exuberant axon arbor growthExuberant axon arbor growth

IN VIVO ROLE IN VIVO ROLE –– ALTERING TARGET TISSUE INFLUENCEALTERING TARGET TISSUE INFLUENCE

Retina Optic TectumBDNFBDNF

TrkBTrkB

P

M

Imaging Imaging presynapticpresynaptic sites in retinal axons sites in retinal axons in vivoin vivo

Yellow Yellow ==GFPGFP--synaptobrevinsynaptobrevin

++ DsRedDsRed((presynapticpresynaptic sites)sites)

GFPGFP--tagged synaptic markers localize to tagged synaptic markers localize to ultrastructurallyultrastructurally identifiedidentifiedsynapses in the tadpole optic synapses in the tadpole optic tectumtectum

AbAb--associatedassociatedgold particlesgold particles

GFPGFP--SybSyb ControlControl

**

**

Postsynaptic endingPostsynaptic ending

PresynapticPresynaptic terminalterminal****

Manipulating BDNF levels in the optic Manipulating BDNF levels in the optic tectumtectumBDNFBDNF or or blocking antibodies to BDNFblocking antibodies to BDNF

BDNFBDNF ororantianti--BDNFBDNF

Time lapseTime lapse

Synapses are formed and eliminated in actively branching axon arSynapses are formed and eliminated in actively branching axon arborsbors

AlsinaAlsina et al., Nature et al., Nature NeurosciNeurosci., 2001., 2001

Analysis of axon branch and Analysis of axon branch and presynapticpresynaptic site dynamicssite dynamics

2 hr

StableStable

AddedAdded

EliminatedEliminated

2 , 4, 6, 8, 24 hr2 , 4, 6, 8, 24 hr

BDNF enhances RGC axon arbor complexity and synaptic numberBDNF enhances RGC axon arbor complexity and synaptic number

ControlControl

BDNFBDNF

AlsinaAlsina et al., Nature et al., Nature NeurosciNeurosci., 2001., 2001

ControlBDNFAnti-BDNF

0-2 hr 0-4 hr 0-6 hr 0-24 hr0

50

100

150

200

250

300

350

Time interval

Bra

nch

Num

ber (

%)

0-2 hr 0-4 hr 0-6 hr 0-24 hr0

50

100

150

200

250

300

350

Time interval

pres

ynap

ticcl

uste

r num

ber (

%)

1 2 34

5

BDNF influences axon arbor branch numberBDNF influences axon arbor branch number……

……and synapse numberand synapse number

**** ******

********

****

****

****** ******

************ pp < 0.05< 0.05

pp < 0.005< 0.005p p < 0.0005< 0.0005

Syna

ptic

clu

ster

nu

mbe

r / a

rbor

leng

th

Time interval

0-2 hr 0-4 hr 0-6 hr 0-24 hr

1.0

1.5

2.0

ControlControl

BDNFBDNF

BDNF increases synaptic BDNF increases synaptic innervationinnervation density per axon terminaldensity per axon terminal

Alsina et al., Nature Neurosci., 2001

ControlControl

p

m

Anti-BDNFAntiAnti--BDNFBDNF

0h0h 2h2h 4h4h

GFP-Syb 10 µm

Neutralizing endogenous BDNF with blocking antibodies promotes Neutralizing endogenous BDNF with blocking antibodies promotes axon branch and synapse eliminationaxon branch and synapse elimination

((HuHu, , NikolakopoulouNikolakopoulou and Cohenand Cohen--Cory, Development 2005)Cory, Development 2005)

Anti-BDNFAnti-BDNF

PresynapticPresynaptic sites are destabilized and eliminated in the absence of BDNFsites are destabilized and eliminated in the absence of BDNF

ControlAnti-BDNF

0-2 2-4 4-6 6-8 8-240

25

50

75

100

Time interval (hours)

Stab

le a

xon

bran

ches

** ** **

0-2 2-4 4-6 6-8 8-240

25

50

75

100

Time interval (hours)

Stab

le p

resy

napt

iccl

uste

rs(%

)

**

******

**** ****

2 hr2 hr

2 hr2 hr

BDNF increases axon arbor complexity by promoting branching BDNF increases axon arbor complexity by promoting branching and growth. and growth.

BDNF increases the density of synaptic BDNF increases the density of synaptic innervationinnervation per axon per axon terminal.terminal.

Neutralizing endogenous BDNF induces synapse disassemblyNeutralizing endogenous BDNF induces synapse disassemblyand axon branch elimination.and axon branch elimination.

TectalTectal neuronsneurons

BDNFBDNFGFPGFP--TrkB.T1TrkB.T1

RFPRFP--synaptobrevinsynaptobrevin(eye (eye lipofectionlipofection))

Are the effects of BDNF direct on Are the effects of BDNF direct on presynapticpresynaptic terminal axons?terminal axons?

Truncated TrkB.T1 acts as a dominant negative receptorTruncated TrkB.T1 acts as a dominant negative receptor

TrkB.FL

TK domainTK domain

TrkB.T1

Signal no signal

GFPGFP--TrkB.T1 expressing optic axons project appropriately to TrkB.T1 expressing optic axons project appropriately to the optic the optic tectumtectum in cellin cell--specific transgenic tadpolesspecific transgenic tadpoles

MarshakMarshak et al., J. Neuroscience 2007et al., J. Neuroscience 2007

GFPGFP-- TrkB.T1TrkB.T1under under AchRAchR promoterpromoter

Transgenic Xenopus embryos Transgenic Xenopus tadpole

GFPGFP--TrkB.T1TrkB.T1GFPGFP--controlcontrol

Altering Altering TrkBTrkB signaling influences axon growth cone morphology signaling influences axon growth cone morphology at the targetat the target

branched

gc

bran

ched

growthcone

GFPGFP--TrkB.T1TrkB.T1GFPGFP--controlcontrol

Branched RGC axons expressing GFPBranched RGC axons expressing GFP--TrkB.T1 retain growth coneTrkB.T1 retain growth cone--like structures and develop few like structures and develop few presynapticpresynaptic sitessites

GFPGFP--controlcontrol GFPGFP--TrkB.T1TrkB.T1

*

RFPRFP--sybsyb RFPRFP--sybsyb

MarshakMarshak et al., J. Neuroscience 2007et al., J. Neuroscience 2007

Expression of GFPExpression of GFP--TrkB.T1 in TrkB.T1 in RGCsRGCs results in simpler axon arborsresults in simpler axon arborswith high branch turnover rates with high branch turnover rates

MarshakMarshak et al., J. Neuroscience 2007et al., J. Neuroscience 2007

StableAddedEliminatedAdded and then eliminated

PresynapticPresynaptic site densitysite density

2h 4h 8h 24h6h

** ** ** ** **

0.00

0.25

0.50

0.75

1.00

1.25

Syna

ptic

den

sity

(clu

ster

s pe

r 10μ

m)

Branch dynamicsBranch dynamicsGFP- Control

GFP-TrkB.T1

Cha

nges

in b

ranc

h nu

mbe

r(e

very

2 h

ours

)

** **

Control

TrkB.T10

20

40

60

80

100

Control

TrkB.T1

StableStable AddedAdded

BDNF/BDNF/TrkBTrkB

RGCsRGCsRGCs

TectalTectal neuronsneurons

Cell autonomous Cell autonomous TrkBTrkB signaling in signaling in RGCsRGCs modulates modulates the establishment of the establishment of retinotectalretinotectal synaptic connectivitysynaptic connectivity

Expression of GFPExpression of GFP--TrkB.T1 in single RGC axons alters TrkB.T1 in single RGC axons alters synaptic vesicle number at synaptic vesicle number at retinotectalretinotectal synapsessynapses

GFPGFP--controlcontrol

GFPGFP--TrkB.T1TrkB.T1

500nm

70.8 70.8 ±± 6.166.16Synaptic vesicle poolSynaptic vesicle pool

48.3 48.3 ±± 5.01 5.01 **Synaptic vesicle poolSynaptic vesicle pool

Docked vesiclesDocked vesicles6.4 6.4 ±± 0.460.46

Docked vesiclesDocked vesicles3.9 3.9 ±± 0.59 0.59 **

Imaging postsynaptic sites in Imaging postsynaptic sites in tectaltectal neuron neuron dendriticdendritic arbors arbors in vivoin vivo

Yellow Yellow ==PSDPSD--9595--GFPGFP++ DsRedDsRed

ControlControl

0 hrs 24 hrsAntiAnti--BDNFBDNF

Blocking endogenous BDNF inducesBlocking endogenous BDNF inducespostsynaptic site eliminationpostsynaptic site elimination

(Sanchez et al, Development, 2006)(Sanchez et al, Development, 2006)

BDNF influences postsynaptic site number by 24 hoursBDNF influences postsynaptic site number by 24 hoursbut does not alter but does not alter dendriticdendritic branchingbranching

PSDPSD--9595--GFP GFP punctapuncta

ControlBDNFAnti-BDNF

0

50

100

150

200

250

300

350

**

**

****

**

Time interval0-4h 0-24h 0-48h

tota

l PSD

95-G

FP p

unct

a(%

of i

nitia

l)

(Sanchez et al, Development, 2006)(Sanchez et al, Development, 2006)

Total branchesTotal branches Total lengthTotal length

0

50

100

150

200

250

300

350

Time interval0-4h 0-24h 0-48h

tota

l bra

nche

s (%

of i

nitia

l)

0

50

100

150

200

250

300

350

**

Time interval0-4h 0-24h 0-48h

tota

l len

gth

(% o

f ini

tial )

BDNF increases synaptic BDNF increases synaptic innervationinnervation density in density in tectaltectalneuron neuron dendriticdendritic terminalsterminals

BDNFControl

0-4 hr 00--24 hr24 hr 0-48 hr

post

syna

ptic

clu

ster

nu

mbe

r / a

rbor

leng

th

Time interval

1.0

1.5

2.0

Postsynaptic Postsynaptic tectaltectalneuronneuron

pres

ynap

ticcl

uste

r nu

mbe

r / a

rbor

leng

th

Time interval

0-2 hr 0-4 hr 00--6 hr6 hr 0-24 hr

1.0

1.5

2.0

PresynapticPresynaptic retinal ganglionretinal ganglioncell axon arborcell axon arbor

TectalTectal neuronsneurons

BDNFBDNF

Imaging Imaging dendriticdendritic morphology and synaptic specializationsmorphology and synaptic specializationsin in tectaltectal neurons with altered neurons with altered TrkBTrkB signalingsignaling

GFPGFP--controlcontrol GFPGFP--TrkB.T1TrkB.T1

MarshakMarshak et al., J. Neuroscience 2007et al., J. Neuroscience 2007

SNAPSNAP--2525immunostainingimmunostaining

GFP-Control

GFP-TrkB.T1

Control TrkB.T10

10

20

Den

drite

mot

ility

rate

(%± s

.e.m

.)

TrkB.T1Control0

1

2

3

Syna

ptic

den

sity

(SN

AP-

25 c

lust

ers

/10μ

m)

0h 24h 48h0

1

2

Den

driti

cco

mpl

exity

inde

x

Altering Altering TrkBTrkB signaling in signaling in tectaltectal neurons does not influenceneurons does not influencedendrite branch dynamics or synapse densitydendrite branch dynamics or synapse density

BDNF

SingleSingle--cell manipulations in cell manipulations in TrkBTrkB signalingsignalingCellCell--autonomous autonomous TrkBTrkB signaling influences axon arbor shape signaling influences axon arbor shape and synaptic site stability. and synaptic site stability. TrkBTrkB--mediated changes at synapses mediated changes at synapses can significantly affect synaptic function (EM observations can significantly affect synaptic function (EM observations ––decreased synaptic vesicle number). decreased synaptic vesicle number).

Confirm antiConfirm anti--BDNF effectsBDNF effects

Support Support BDNFBDNF’’ss potential to rescue developing circuits with potential to rescue developing circuits with altered activity or after injuryaltered activity or after injury

Sonia Sonia MarshakMarshakMargarita Margarita MeynardMeynardMariangelaMariangela NikolakopoulouNikolakopoulou

Supported by:Supported by:National Eye InstituteNational Eye Institute

BertaBerta AlsinaAlsinaThuyThuy VuVuBing Bing HuHuAnalizaAnaliza SanchezSanchezSanaSana JavedJavedBen MatthewsBen Matthews