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Supporting Online Material for

Input-Specific Spine Entry of Soma-Derived Vesl-1S Protein Conforms to

Synaptic Tagging

Daisuke Okada,* Fumiko Ozawa, Kaoru Inokuchi*

*To whom correspondence should be addressed. E-mail: [email protected] (K.I.); [email protected] (D.O.)

Published 15 May 2009, Science 324, 904 (2009) DOI: 10.1126/science.1171498

This PDF file includes: Materials and Methods

SOM Text

Figs. S1 to S10

Tables S1 to S3

References and Notes

SOM: Okada, Ozawa & Inokuchi

2

Materials and Methods

Plasmids and neuron culture

The V1p (vesl-1 minimal promoter)-Vesl-1S-EGFP (VE) and V1p-EGFP plasmids

were constructed from our differential display library (mouse V1p-Vesl-1S1SpA in pGL3

vector) (S1, S2) and pEGFP-N2. V1p-Vesl-1S-PAGFP was constructed from VE and

PAGFP. pDsRed2-N1 was purchased from Takara Bio. VW24AE was prepared by insertion

of the W24A fragment generated by polymerase chain reaction. Hippocampal neuron

culture was prepared from E18 Wistar rat embryos on glass coverslips and transfected on

the 8th or 9th day in vitro (DIV 8-9) as previously reported (S2). DsRed2, EGFP and VE

proteins were evenly expressed in a small proportion of neurons throughout a neuron

including spines on DIV 18-25.

Dual luciferase assay.

A hippocampal culture was transfected with the vesl-1 minimal promoter-driven firefly

luciferase (V1p-GL3) and the thymidine kinase promoter-driven Renilla reniformis

luciferace (pTK-RL) (S1). On the following day, cells were incubated with 20 mM forskolin

and 0.1 mM IBMX (FI) for 20 min, in the presence or absence of 20 mM CHX. Cells were

collected after 3 h. The luciferase activity ratio (GL/RL) was calculated in triplicate or


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quadruplicate. Increase ratio relative to no-stimulus control (NoStm) are calculated.

In situ hybridization of hippocampus of in vivo LTP rats

In situ hybridization was performed with rat hippocampal slices prepared 2 h after

stimulation at the medial perforant path which evoked late-phase long-term potentiation in

vivo (S3, S4).

Image acquisition

On DIV 21-28, neurons cultured on a glass coverslip were placed in a recording

chamber and received constant perfusion at 27.0 ± 0.5°C (TC-344B temperature controller)

at a rate of 0.5 ml/min using a peristaltic pump throughout the experiment. Normal ACSF

contained (in mM) NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1.5, NaH2PO3 1, NaHCO3 25,

glucose 10 and bicuculin methiodide 0.05, and constantly infused with 5% CO2 and 95%

O2. Mg-free ACSF did not contain MgCl2 , and the CaCl2 concentration was reduced to 2.0

mM to avoid morphological disturbances.

For bath application of various reagents, cells were set in a perfusion chamber.

Fluorescence images were acquired using an upright microscope equipped with a

water-immersion objective (60´, numerical aperture 0.90), a cooled CCD camera (1392 ´

1040 pixels), a 100 W mercury lamp, a 6 % neutral density filter, a shutter (Mac5000 filter


4

wheel) and a Z-axis motor. For microperfusion experiments, we used an inverted

fluorescence microscope, an EM-CCD camera (512 ´ 512 pixels), a 100 W mercury lamp,

a 10 % neutral density filter, a shutter, a Z-axis motor and a hand-made acrylic chamber.

For photoactivation, a Micropoint ablation system attached to the microscope was used.

Fluorescence of Stillbene420 (404 nm) excited by the N2 laser was focused through the

objective lens (63´, numerical aperture 1.40). The entire cell body of a neuron was

illuminated at 20 Hz for 5 sec by manual scan. Appropriate filter sets were used to observe

the fluorescence signals for VE, EGFP, VPA and PAGFP (exitation 470/20 nm; dichroic

mirror 493 nm; emission 505-530 nm), DsRed2 and rhodamine (565/30; 585; 620/60), and

AlexaFluor633 (630/20; 649; 667/30). Free AlexaFluor633 was prepared by hydrolysis of

AlexaFluor633 carboxylic acid succinimidyl ester in 1 M Tris at pH8.5 and was used

after filtration. VE, VPA, EGFP and PAGFP were observed 3 times every 10 min before

stimulation and every 15-30 min after stimulation. VPA was photoactivated in the soma of

DsRed2-expressing neurons, the “before” images were captured and then the cell was

stimulated by microperfusion. The interval between photoactivation and microperfusion

was usually 15-25 min. The “after” image was acquired 240 min after the onset of

microperfusion. The acquisition and analysis of images were performed using the


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MetaMorph software. Images were stored in the HDD of a personal computer for analysis

off line.

Microperfusion

For microperfusion, two kinds of borosilicate glass pipettes (outer diameter 1 mm)

were prepared by a puller. An injector electrode (tip diameter ~5 mm; patch pipette-like

shape) was set near the target cell at a 45o angle, while another electrode for suction (tip

diameter ~20 mm; straight shank, the tip beveled at 45o angle using an EG6 beveller) was

set in an opposed position at 135o angle, using MP330 manipulators. The injection pipette

was connected to a pressure injector via a reservoir. The entire line between the reservoir

and the pipette was filled with ACSF containing reagents and rhodamine or AlexaFluor633

(for DsRed2-containing cells), which was ejected by impressing enough pressure to

overcome the capillary action. Ejection was stable throughout the microperfusion (10 min).

Image analysis

Z-series images from 3-5 different focal planes with 0.5 mm intervals were taken at

each time point. A bird’s eye image was reconstructed in which the intensity information in

the observed thickness was maintained intact. A stack of bird’s eye images covering an

entire experiment was obtained and movements along the X and Y axes were cancelled by


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the alignment of each image. Regions of interest (ROIs) wrapping around each spine head

were selected and the average signal intensity of each ROI was measured. An additional 5

ROIs were selected on the background (without fluorescent structure) and their average

intensity was subtracted from spine intensity values. Spines at the proximity of the first

dendritic branching, filopodia and stubby spines, and spines that appeared or disappeared

during experiments were excluded from the analysis.

A line scan was performed to estimate the peak intensity of spine VE and spine

dimension. A line parallel to the base dendrite was taken in a spine, along which intensity

was scanned. The maximal value along the line after subtraction of background intensity

was set as the peak intensity. There were two points along the line where the intensity was

half that of the peak. The distance between them was set as the half-width of the spine,

which is representative of spine size.

Bath application experiments were conducted with proper within-lot controls and

significance was calculated by t test of the final trap index (240 min after the onset of

stimulation) between groups. The frequency of the increase in spine fluorescence over the

pre-stimulus average (F/Fpre) empirically matched a normal distribution. We selected a

representative F/Fpre falling on the average + S.D., which appears at a cumulative frequency


7

of 0.8413 in the normal distribution (trap index). The trap index consistently evaluates the

rightward shift of the cumulative curves (Fig. 1); however it requires 100 or more spines

for analysis.

In the microperfusion experiments regions inside and outside the perfusion area

were separately analyzed and compared; thus, the number of spines was too small to

calculate the trap index reliably. In this way, we applied a c2 test first to estimate whether

F/Fpre follows a normal distribution. When c2 test detects significant deviation from normal

distribution due to large outliers, the deviation was attributed to VE trapping in some spine.

When c2 test estimated the F/Fpre ensemble as a normal distribution, we applied a t test to

estimate the significance of F/Fpre difference between inside and outside the perfusion area.

Increases of VPA fluorescence (initially zero) were calculated by the measurement

of fluorescence intensity using green fluorescence filters, which bleed through 0.5 % of

DsRed fluorescence. DsRed2 fluorescence did not show any trapping thereby not affecting

the VPA trapping analysis; however, this caused an underestimation of VPA changes.


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Supporting Discussion

1. Choice of the monitoring PRP

Late-phase synaptic plasticity depends on the function of newly synthesized PRPs in

activated spines. PRP synthesized in soma should enter spines before being integrated as

postsynaptic components or modulators. If the spine entry of a PRP is regulated by synaptic

input, it may serve as a synaptic tag. This working hypothesis was examined using an

EGFP-fused PRP, with the PRP synthesized in the soma in response to synaptic input. The

monitoring PRP should not be synthesized in the dendrite as is the case for PKMz (S5) and

GluR1 (S6). The expression of the monitoring PRP in spines should be altered only in the

late-phase, but not in the early-phase as is the case for GluR1 (S7). We therefore selected

Vesl-1S, which is a short-form variant of the Vesl family of proteins, as the monitoring

protein (S8, S9). Vesl-1S is rapidly induced in the soma by late-phase long-term

potentiation (S10, S11) (Fig S5), and transiently functions in synapses by counteracting the

scaffolding function of the Vesl long-form, which leads to synaptic rearrangements (S12)

and long-term memory (S13), as well as to the regulation of the agonist-independent

activation of mGluRs (S14) and of ryanodine receptor functions (S15).


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2. Neuronal mechanisms of VE trapping in culture

In the dispersed neuron culture, axonal branches find their targets and make

random connections during development in vitro. Cells spontaneously fire, thus axons

spontaneously release various factors including glutamate and probably the

TTX-sensitive factor (Fig.6). Every spine is supposed to have different magnitude and

probability of inputs from neuron networks in the dish. Mg-free ACSF caused synaptic

NMDA receptor activation via glutamate thus released from presynaptic sites (S16).

Therefore, probability and magnitude of VE trapping are variable among spines.

High concentrations (1 mM) of TTX suppresses spike propagation in both

dendrites and axons, while low concentrations (10-50 nM) of TTX suppress only

back-propagating Na+ spikes in the dendrites in both hippocampal slices and primary

culture (S17). This distinct TTX-sensitivity is explained by different Na+ channel

density in the axons and the dendrites in these preparations. TTX at 1 mM suppressed

VE trapping after PKG activation, while TTX at 50 nM did not (Fig.6A), suggesting

that VE trapping require spike propagation in presynaptic fibers but not in the dendrites.

Thus, VE trapping requires synergistic activation of both sides of synapses;

presynaptic release of glutamate and TTX-sensitive factors, and postsynaptic NMDA


10

receptors.

3. Other candidate molecules involved in synaptic tagging (Table S3)

Table S3 shows trap index averages (+ S.D.) 240 min after the onset of Mg-free

ACSF, to summaries effects of inhibitors for mGluR1/5, PKA, PKCa/b2 and TrkB. These

inhibitors had no effects by themselves. Among them, PKA is of special interest, because it

has been implicated to be selectively involved in late-phase plasticity (S18, S19). Although

our data show that PKA is not involved in VE trapping, multiple roles of PKA in synaptic

tagging and late-phase plasticity were suggested by electrophysiological measurements

(S20, S21).

Previous works have also identified other PRP candidates, such as PKMz (S22),

Ca-calmodulin-dependent kinases and MEK1/2 (S23). Although we tested effects of 10 mM

KN93 (Ca-calmodulin-dependent kinases), 1 mM MyrZIP (PKMz) and 1 mM U0120

(MEK1/2), they resulted in severe morphological changes in dendrites and spines of

cultured neurons, which prevented VE trap measurements.


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4. On the persistence of VE trapping activation

Figure 6 shows that the PKG or its downstream activities are persistently active with

a lifetime of less than 4h. This persistence of VE trapping suggests a mechanism

advantageous for the recruitment of multiple PRPs that arrive from the soma at various

times. However, the observed persistence was somewhat longer than that reported for

associative late-phase long-term potentiation in slices (S24), and far longer than that

reported in vivo (S19). These discrepancies may be partly explained by the lower

temperature used in our experiments. We further imply that multiple steps with distinct

lifetimes are involved in late-phase plasticity; therefore protein entry and

electrophysiological responses may have different lifetimes. The persistence of VE trapping

seems to depend on the presence of presynaptic inputs: although the constant presence of

TTX abrogated VE trapping 4 h after PKG activation (Fig. 6C), VE trapping was active

even 6 h after PKG activation (Fig. 6B), when the neuron received TTX only for 2 h.

Electrophysiological experiments also have shown that the lifetime of synaptic tagging is

variable (S25). These results suggest that integrity of the preparation used in the

experiments also affects persistence of tagging.


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5. Synaptic tagging and multiple PRP sorting

Late-phase plasticity requires induction of expression of multiple genes (S26), and

includes F-actin modification in the spine (S2, S27). VE trapping was not accompanied by

prominent changes in spine size, nor was it correlated with initial spine size (Fig. 1E),

which suggests that entry of a single kind of PRP does not necessarily affect morphological

plasticity. Thus, we propose that synaptic tagging involves multiple mechanisms that are

specific to individual PRPs. These mechanisms may work synergistically or cooperatively

to achieve persistent late-phase plasticity. For example, activity-dependent regulation of the

structure and function of the postsynaptic protein complex is another candidate for synaptic

tagging (S18-S23). PRPs during the dendritic transport from soma are subject to VE

trapping (fig.S9). Transport vesicles are composed of multiple cargos, adaptor and motor

proteins (S28), thereby suggesting that PRPs move and function as a group. These groups

of PRPs may have common and specific components, which may be advantageous to

achieve cross-tagging, which predicts common molecular mechanisms for synaptic tagging

of long-term potentiation and depression (S22,S23).

Recently, idea of clustered plasticity, or plasticity taking place in dendritic


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compartment, has been proposed (S29). Our observation that NO is involved in VE

trapping (fig. S10) suggests that the diffusion-based NO spread may contribute to synaptic

tagging of a compartment. However, it is noted that VE trapping is not fully activated when

NO activates local PKG in a compartment. VE trapping may be active at the spine where

NMDA receptor is activated, while surrounding spines where PKG was activated by NO

travelled from the active spine are in a primed state for trapping. A next input with the

TTX-sensitive factor during persistent activity of PKG (or downstream) is required for VE

trapping at these spines. This may be advantageous for integration of information, the basis

of associative memory.

A

B

Cfunction

incorporationPRP entry

late-phase-evoking activity

early-phase-evoking activity

no activity

induction

synthesis

soma

soma

soma

tagged spine

new PRP

dendritic transport

spine(not tagged)

tagged spine

late-phase plasticity(input-specific)

late-phase plasticity(associative)

no plasticity

Okada et al. Fig.S1

60

40

20

0

VE EGFP

EGFP

VE

A B

Okada et al. Fig.S2

A B C

before

after

Okada et al. Fig.S3

CHXFI No Stm

incr

ease

(fold

)

10

0

20

30

40

50

Okada et al. Fig.S4

A B

Okada et al. Fig S5

before after subtraction

INSIDE

INSIDE

DsRed2 VPA

OUTSIDE

Okada et al. Fig.S6

F/Fpre (VPA)

F/Fp

re(D

sR)

2

1

00 1 2 3

VPA-NMDA/Gly/Mg-free

2

1

00 1 2

VPA-ACSF

A B

3

F/Fp

re(D

sR)

F/Fpre (VPA)

Okada et al. Fig.S7

final

F/Fp

re

2

0

INSIDE OUTSIDEPAGFP

t test P= 0.81

2

0

OUTSIDEINSIDEDsRed2

t test P= 0.21

PAGFPNMDA/Gly/Mg-free

A B

final

F/Fp

re

Okada et al. F.igS8

final

F/F pr

efin

alF/

F pre

2

0

2

0

INSIDE

OUTSIDEINSIDE

OUTSIDEVPA

DsRed2

t test P= 0.955

t test P= 0.505

NMDA/Gly/Mg-free+colchicine

A

B

Trap

Inde

x

2.5

2

1.5

1

0.5

00 60 120 180 240

min

a b

Okada et al. Fig.S9

A2.5

2

1.5

1

0.5

00 60 120 180 240 min

L-NAME

Trap

Inde

x

B

2

1

00 60 120 180 240 min

3

Fe-DTCS

trap

inde

x

D

0 60 120 180 240 min

2.5

2

1.5

1

0.5

0

Rp-8BrPET-cGMPS

trap

inde

x

FTr

apIn

dex

ODQ

0

0.5

1

1.5

2

2.5

0 60 120 180 240 min

NO donors

0 60 120 180 240 min

2.5

2

1.5

1

0.5

0

Trap

Inde

x

C

E

Trap

Inde

x

0

0.5

1

1.5

2

2.5

0 60 120 180 240 min

Ca=0 +EGTA

G

80

0

µm F/Fp

re

t-test p= 0.8940

1

2INSIDE OUTSIDE

spines

NMDA/Gly/Mgfree +ODQ

Okada et al. Fig.S10


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Fig. S1. Synaptic tagging hypothesis

To explain the role of somatically synthesized PRPs in input-specific late-phase plasticity,

three hypotheses were originally proposed (S30). The local synthesis hypothesis assumes

that PRPs are synthesized locally from mRNAs in dendrites near the activated spines (S31).

Although some PRPs are recruited by this mechanism (S5, S6), other PRPs are synthesized

in the soma, which is not explained by the local synthesis. The mail hypothesis assumes

that PRPs newly synthesized in the soma are marked for their destination synapse, which

was excluded by the observation of associative late-phase long-term potentiation in the

two-pathway experiments (S30). Finally, a synaptic tag is assumed in the synapse that

receives the plasticity-evoking activity. According to the synaptic tagging hypothesis, new

PRPs synthesized in the soma are transferred in dendrites over the entire cell without any

pre-determined destination. These PRPs are thought to function only in tagged synapses.

This concept of synaptic tagging (S30) has been supported by a number of experiments

(S18-S25, S32-S34). This figure schematically depicts the characteristics of synaptic

tagging. A: Neuronal activity evoking late-phase plasticity, but not early-phase, triggers the

somatic synthesis of new PRPs. B: New PRPs (irradiating circles: ) are carried over the

entire neuron via the dendritic transport (red arrows). On the other hand, activities evoking


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both late- and early-phases of plasticity mark spines (synapses) with a synaptic tag during a

limited lifetime via an NMDA receptor-dependent mechanism. Synaptic tagging itself is

independent of PRP synthesis, including local synthesis. C: Our hypothesis. PRPs can

function only in the tagged synapses. PRPs should enter spine and be incorporated into

postsynaptic structures before the onset of their functions (blue arrow); thus, tagged spines

that received either early- or late-phase evoking activity exhibit input-specific or

associative late-phase plasticity, respectively. Spines that do not receive plasticity-evoking

activity are not tagged and do not exhibit plastic changes.

Fig. S2. Spine density of VE- and EGFP-expressing neurons

A: Inverted black-and-white images of VE and EGFP fluorescence in neurons. Bar = 10 mm.

B: Spines were visualized by co-transfection of DsRed2 with VE or EGFP. The number of

spines along a dendrite of 100 mm was counted in cells from 3 different cultures. Average

spine numbers of 47 EGFP-expressing cells (41.7 ± 18.5 (S.D.)) and 31 VE-expressing

cells (34.5 ± 16.2) were not significantly different (P= 0.085, t test). These results indicate

that both fluorescent proteins were distributed evenly within neurons and that expression of

VE did not affect the spine density.


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Fig. S3. Input-specific VE trapping

Close-up images of spines before and 4 h after microperfusion with Mg-free ACSF

containing NMDA and glycine are shown in black-and-white and pseudocolors. Bars: 0.5

mm. A: Spines inside the microperfusion area showing VE trapping. The F/Fpre values were

2.36 and 2.31. B: Spines inside the microperfusion area not showing VE trapping. The

F/Fpre value was 1.22. C: Spines outside the microperfusion area. The F/Fpre values were

1.18 and 0.91.

Fig. S4. Dual luciferase assay

In our plasmid, VE expression was regulated by the vesl-1 gene minimal promoter (V1p),

which contains 4 CREs essential for induction of expression (S1). We confirmed that the

V1p promoter was activated by a mixture of forskolin and 3-isobuthyl 1-methyl xanthine

(IBMX) (FI, bath application) using a dual luciferase assay. Mg-free ACSF had no effect on

the activity of the V1p promoter (unpublished observation by Niibori, Okada and Inokuchi).

Increase ratio in the luciferase activity ratio (GL/RL) relative to no-stimulus control

(NoStm) are shown. Vertical bars represent S.D. FI significantly induced expression of

V1p-GL3 (t-test p = 0.003), while it failed to do so in the presence of CHX (P = 0.49, t


27

test).

Fig. S5. In situ hybridization of Vesl-1S mRNA in hippocampus

Late-phase LTP was evoked by stimulating the entorhinal cortex 2 h before decapitation for

in situ hybridization as described in (S3). (A) Hippocampus under a 4x objective, bar: 500

mm. (B) Boxed region in (A) magnified under a 20x objective, bar: 100 mm.

Fig. S6. Other examples of VPA trapping by NMDA receptor activation

Black-and-white images of DsRed2 (initial) and VPA (before and 240 min after

microperfusion), and pseudocolour images of the VPA subtraction (after-before) of 2 spines

inside (F/Fpre of VPA = 3.04 and 2.67) and 1 outside (F/Fpre of VPA = 1.31) the

microperfusion area. Bar: 1 mm.

Fig. S7. Correlation between final F/Fpre of VPA and DsRed2

F/Fpre of VPA or DsRed2 obtained 240 min after stimulation in all cells (see Table S2) was

pooled and their correlation was calculated. A: NMDA receptor was stimulated locally by

microperfusion. The correlation coefficient for inside spines (magenta points and the red


28

regression line) was r = 0.11, while that for outside spines (blue points and the black

regression line) was r = 0.46. B: Correlation coefficients of inside and outside spines after

microperfusion with normal ACSF were 0.44 and 0.38, respectively. The small r obtained

for spines inside the stimulated area can be explained by VPA trapping without DsRed2

increase.

Fig. S8. PAGFP did not respond to NMDA receptor activation

Unlike VPA, PAGFP was not trapped by microperfusion with Mg-free ACSF containing

NMDA and glycine. See the legends to Fig. 4 for details and Table S2 for a summary of

results.

Fig. S9. Disruption of vesicular transport affected VE trapping

A: Trap index time-course analysis of bath-perfusion with Mg-free ACSF (red horizontal

bar) showed that the W24A mutant was not trapped in spines. Wild-type VE ( ), V W24A E

( ) and ACSF alone (VE) ( ). B: Colchicine disrupted VPA trapping via local NMDA

receptor activation. Extracellular medium contained 1 mM colchicine throughout the

experiment (from 1 h before to 4 h after microperfusion with Mg-free ACSF containing


29

NMDA and glycine). See the legend to Fig. 4 for details and Table S2 for the summary of

results.

Fig. S10. Downstream of NMDA receptor activation

All panels of this Figure, with the exception of G, depict trap index time-course analysis of

experiments with bath perfusion stimulation. Mg-free ( , N = 7), ACSF ( , N = 5),

Mg-free + reagent ( ), reagent alone ( ), or NO donor ( ). Rectangles show application

of Mg-free ACSF (red), NO donor (grey) or other reagents (blue). A: The omission of

extracellular Ca2+ ions accompanied by a further depletion via 1 mM EGTA led to an

absence of VE trapping stimulated by Mg-free ACSF (N = 4; at 240 min; P = 3.5´10-4 vs

Mg-free, t test). B: A specific inhibitor of NO synthase (10 mM Nw-nitro-L-arginine

methylester; L-NAME) blocked the Mg-free ACSF-dependent VE trapping (N = 5; P =

2.6´10-4, t test). C: NO donors (100 mM 1-hydroxy-2-oxo-3-(N-methyl-

3-aminopropyl)-3-methyl-1-triazene; NOC7 or 200 mM S-nitroso-N-acetylpenicillamine;

SNAP) evoked VE trapping (N=6; P= 1.4´10-3 vs ACSF, t test). D: The scavenging of

extracellular NO using 10 mM of Fe-DTCS (N-(dithiocarboxy)-sarcosine) complex (S35), a

water-soluble NO scavenger which eliminates extracellular NO without affecting


30

intracellular NO (S36), did not affect the Mg-free-ACSF-dependent VE trapping (N = 3; P

= 0.96, t test), suggesting an essential role for NO as an intracellular messenger for VE

trapping. E: A selective inhibitor of soluble guanylyl cyclase, 10 mM

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibited the VE trapping induced by

bath perfusion with Mg-free ACSF (N = 5; P = 2.0´10-4, t test). F: A specific PKG inhibitor,

1 mM Rp-8-bromo-b-phenyl-1,N²-ethenoguanosine-3',5'-cyclic monophosphorothioate

(Rp-8BrPET-cGMPS), blocked the Mg-free ACSF-dependent VE trapping (N = 5; P =

3.9´10-4, t test). G: Microperfusion with Mg-free ACSF containing NMDA and glycine is

likely to evoke VE trapping through the same signalling pathway, because ODQ at 10 mM

also inhibited the VE trapping induced by microperfusion (N = 4). For microperfusion

methods see the legend to Fig. 2, and Table S1 for the summary of results. The white

horizontal bar represents 10 mm.

Table S1Statistics of Microperfusion Experiments (Fig.2)

NMDA/Glycine/Mg-free ACSF

spines ave SD c2-test p spines ave SD c2-test p t-test pcell 1 38 1.09 0.27 3E-05 13 1.00 0.12 0.68cell 2 23 1.38 0.45 1E-16 37 1.13 0.20 1.00cell 3 41 1.60 0.35 3.6E-03 108 1.36 0.20 0.11total 102 158cell 4 19 1.27 0.19 0.62 43 1.07 0.14 0.34 3.0E-04cell 5 29 1.21 0.22 0.30 20 0.95 0.20 0.82 0.03cell 6 38 1.28 0.33 0.28 86 1.07 0.25 0.21 7.3E-04cell 7 30 1.28 0.24 0.37 61 1.09 0.21 0.33 2.8E-04cell 8 29 1.11 0.18 0.60 44 0.97 0.11 0.90 1.3E-04total 145 254grand total 247 412

nACSF

spines ave SD c2-test p spines ave SD c2-test p t-test pcell 9 36 1.20 0.17 0.66 24 1.22 0.29 0.70 0.72cell 10 20 1.20 0.25 0.69 31 1.18 0.28 0.11 0.84cell 11 14 1.05 0.40 0.45 19 0.86 0.52 0.08 0.25cell 12 19 1.04 0.27 0.87 20 1.04 0.27 0.27 0.96cell 13 20 1.11 0.08 0.98 21 1.15 0.15 0.17 0.32total 109 115

NMDA+MK801

spines ave SD c2-test p spines ave SD c2-test p t-test pcell 14 29 1.14 0.22 0.66 42 1.07 0.21 0.37 0.18cell 15 25 1.12 0.17 0.85 39 1.06 0.14 0.69 0.16cell 16 42 1.01 0.15 0.93 27 1.00 0.20 0.54 0.83total 96 108

NMDA+MK801then Mg-free ACSF

spines ave SD c2-test p spines ave SD c2-test p t-test pcell 17 42 0.98 0.15 0.93 43 1.09 0.28 0.26 0.03cell 18 29 1.18 0.20 0.66 40 1.17 0.34 8.4E-07cell 19 24 1.02 0.14 0.55 17 1.28 0.48 6.0E-11total 95 100

NMDA+ODQ

spines ave SD c2-test p spines ave SD c2-test p t-test pcell 20 18 0.99 0.12 0.98 19 0.91 0.22 0.60 0.18cell 21 57 1.21 0.24 0.08 94 1.21 0.29 0.11 0.89cell 22 59 1.13 0.20 0.61 69 1.07 0.21 1.00 0.09cell 23 53 1.03 0.27 0.78 35 1.02 0.25 0.51 0.85total 187 217

INSIDE OUTSIDE

INSIDE OUTSIDE

INSIDE OUTSIDE

INSIDE OUTSIDE

INSIDE OUTSIDE

Table　S2Statistics of VPA Experiments (Figs. 4 and 5)

VPA,NMDA/Glycine/Mg-free ACSFVPA DsRed2

spines ave SD c2-test p spines ave SD c2-test p t-test p spines ave SD c2-test p spines ave SD c2-test p t-test pcell 1 18 1.34 0.38 0.04 14 1.39 0.19 0.54 16 1.75 0.24 0.15 14 1.79 0.25 0.09 0.59cell 2 17 1.55 0.59 8.5E-03 16 1.08 0.18 0.48 17 1.16 0.31 0.64 16 1.16 0.43 0.26 0.99total 35 30 33 30cell 3 13 1.74 0.41 0.435 10 0.82 0.22 0.61 2.0E-05 9 1.61 0.38 0.84 17 1.37 0.29 0.29 0.10cell 4 16 1.41 0.30 0.661 28 1.19 0.23 0.33 8.9E-03 16 1.24 0.38 0.40 28 1.08 0.22 0.79 0.07cell 5 13 1.50 0.58 0.776 21 1.18 0.21 0.53 0.03 11 1.52 0.41 0.46 21 1.40 0.43 0.85 0.46cell 6 31 1.33 0.40 0.167 12 1.06 0.28 0.63 0.04 31 0.91 0.19 0.25 19 0.84 0.16 0.59 0.18

total 73 71 67 85grand total 108 101 100 115

VPA,nACSFVPA DsRed2

spines ave SD c2-test p spines ave SD c2-test p t-test p spines ave SD c2-test p spines ave SD c2-test p t-test pcell 7 33 1.153 0.288 0.340 43 1.047 0.244 0.256 0.087 33 1.183 0.279 0.381 43 1.123 0.259 0.950 0.342cell 8 19 0.967 0.092 0.999 8 0.951 0.075 0.984 0.662 21 1.411 0.131 0.251 17 1.306 0.253 0.546 0.126cell 9 10 0.972 0.148 0.322 12 0.958 0.159 0.488 0.824 10 0.886 0.174 0.391 14 0.866 0.140 0.746 0.140cell 10 19 0.917 0.153 0.995 16 1.039 0.308 0.435 0.138 19 0.971 0.280 0.434 19 0.936 0.290 0.070 0.704total 81 79 83 93

PAGFP,NMDA/Glycine/Mg-free ACSFPAGFP DsRed2

spines ave SD c2-test p spines ave SD c2-test p t-test p spines ave SD c2-test p spines ave SD c2-test p t-test pcell 11 17 1.02 0.11 0.38 27 0.95 0.13 0.13 0.07 17 1.02 0.12 0.32 27 0.94 0.17 0.06 0.11cell 12 38 1.07 0.22 0.85 38 1.01 0.19 0.31 0.17 33 0.83 0.13 0.07 41 0.77 0.17 0.69 0.12cell 13 20 1.00 0.20 0.71 10 0.89 0.17 0.57 0.10 NA NAcell 14 25 0.92 0.28 0.18 26 0.90 0.31 0.24 0.81 25 1.22 0.44 0.47 26 1.09 0.27 0.41 0.21total 100 101 75 94

VPA,Colchicine+NMDA/Glycine/Mg-free ACSFVPA DsRed2

spines ave SD c2-test p spines ave SD c2-test p t-test p spines ave SD c2-test p spines ave SD c2-test p t-test pcell 15 20 1.18 0.20 0.52 23 1.15 0.17 0.77 0.51 20 0.95 0.20 0.18 29 0.95 0.25 0.88 0.96cell 16 8 1.05 0.09 0.21 13 1.12 0.29 0.95 0.52 6 1.12 0.14 0.87 17 1.03 0.22 0.30 0.35cell 17 8 1.20 0.28 0.78 23 1.37 0.37 0.96 0.24 13 1.09 0.36 0.11 25 1.03 0.27 0.93 0.59total 36 59 39 71

NA: Not analyzed

INSIDE OUTSIDE INSIDE OUTSIDE




Table S3 Effects of various inhibitors on Mg-free ACSF-dependent VE trapping Inhibitor (target) Trap index N (cells) t-test p (vs. Mg-free) 33 mM CPCCOEt (mGluR1) +0.3 mM MPEP (mGluR5) 1.91+0.09 3 0.28 400 nM PKAI (PKA) 2.24+0.23 4 0.31 30 nM GF109203X(PKC a/b2) 1.74+0.13 4 0.03 200 nM K252a (TrkB) 1.58+0.14 3 0.01 See also Supporting Discussion 3.

34

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