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					Supplementary information




Early Presynaptic Changes During Plasticity In Cultured Hippocampal
Neurons
Ipe Ninan, Shumin Liu, Daniel Rabinowitz, Ottavio Arancio

Results

An earlier study has demonstrated an increase in number of presynaptic (prS)

boutons (btns) after activation of cAMP signaling in cultured hippocampal

neurons (Ma et al., 1999). Therefore, it was interesting to test whether elevating

cAMP levels by application of forskolin (activates adenylyl cyclase resulting in

increased cAMP) or IBMX (1-Methyl-3-isobutylxanthine, a phosphodiesterase

inhibitor that elevates cAMP) modifies neurotransmitter release in our system.

First, we have tested whether perfusion of forskolin or IBMX increases number of

FM stainable VAMP2 puncta. Perfusion of forskolin (50 M) for 15 min revealed a

time-dependent increase in number of FM stained VAMP2-GFP puncta [15 min

after perfusion: 153.9  7.1% (n=6); 45 min: 168.5  10.3% (n=6); 105 min: 205.9

 12.8% (n=6), F(4,20) = 21.5, P<0.001, one way ANOVA] (Supplementary

Figure S4B). Similarly, perfusion of IBMX (50 M) for 15 min revealed a time-

dependent increase in number of FM stained VAMP2-GFP puncta [15 min after

perfusion: 148.8  5.5% (n=6); 45 min: 153.4  4.5% (n=6); 105 min: 208.2 

7.2%, (n=6), F(4,20) = 15.7, P<0.001, one way ANOVA] (Supplementary Figure

S4B). Next, we examined whether application of tetanus occludes the effect of

forskolin or IBMX on number of functional btns. Tetanus significantly increased


                                                                                1
the number of FM4-64 stained VAMP2-GFP btns (198.2  6.6%, 195.4  4.8%,

202.3  11.6%, and 193.4  11.5%, respectively at 15, 55, 85 and 145 min after

the tetanus, F(4,20) = 50.4, P<0.001, one way ANOVA, Supplementary Figure

S4A and B). However, when forskolin or IBMX were perfused for 15 min after the

tetanus, there was no further change in number of FM4-64 stained VAMP2-GFP

btns [200.1  9.8%, 198.7  8.2%, 193.3  4.9%, and 199.5  7.8%, respectively

at 15, 55, 85 and 145 min after the tetanus (for the tetanus + forskolin group,

P>0.05, one way ANOVA), 197.2  6.9%, 191.7  6.7%, 191.3  6.9%, and 186.4

 5.5%, respectively at 15, 55, 85 and 145 min after the tetanus (for the tetanus +

IBMX group, P>0.05, one way ANOVA, Supplementary Figure S4A and B)]. We

also examined whether forskolin or IBMX affect releasable fluorescence in pre-

existing prS btns. Forskolin revealed an increase in releasable fluorescence in

pre-existing btns in a time-dependent fashion [15 min after perfusion; 137.1 

13.6% (n = 30), 45 min; 147.5  14.1% (n = 30), 105 min; 243.2  23.2% (n=30),

F(4,116) = 30.8, P<0.001, one way ANOVA] (Supplementary Figure S4C).

Similarly, IBMX demonstrated an increase in releasable fluorescence in pre-

existing btns in a time-dependent fashion [15 min after perfusion; 140.6  7.4%

(n=33), 45 min; 156.4  11.3% (n=33), 105 min; 236.7  34.9% (n=33), F(4,128)

= 23.1, P<0.001, one way ANOVA]          (Supplementary Figure S4C). Next, we

examined whether tetanus occludes the effect of forskolin or IBMX on releasable

fluorescence. Application of tetanus significantly increased FM4-64 releasable

fluorescence (194.4  8.6%, 195.2  8.3%, 189.2  8.3%, and 194.5  8.5%,

respectively at 15, 55, 85 and 145 min after the tetanus, F(4,128) = 66.5,


                                                                                 2
P<0.001, one way ANOVA, Supplementary Figure S4A and C). However, when

forskolin or IBMX was perfused for 15 min after the tetanus, there was no further

change in releasable fluorescence [194.2  24.2%, 192.2  21%, 190.3  19.3%,

and 232.3  32.3%, respectively at 15, 55, 85 and 145 min after the tetanus (for

the tetanus + forskolin group, P>0.05, one way ANOVA), 189.3  20.2%, 188.1 

20.6%, 193.2  21.6%, and 222.3  27.4%, respectively at 15, 55, 85 and 145

min after the tetanus (for the tetanus + IBMX group, P>0.05, one way ANOVA,

Supplementary Figure S4A and C)]. The occlusion of the effect of forskolin and

IBMX on number of btns and fluorescence intensity by tetanus suggest that these

two plasticity processes share similar mechanisms.

Discussion

Previous studies have suggested the retrograde messenger NO increases prS

btn number via the NO-cGK-CaMKII pathway (Ninan and Arancio, 2004).

Therefore, in the present study we linked our findings on prS changes occurring

during plasticity with the NO-initiated pathway. We repeated the same

experimental approach using inhibitors of the NO-cGK-CaMKII pathway during

tetanus and studied their effects on different phenomena associated with

synaptic plasticity (SP) including number and releasable fluorescence intensity of

releasing sites, as well as frequency and amplitude of sEPSCs. Both FM4-64

staining and electrophysiological measurements confirmed that the tetanus-

induced SP in cultured hippocampal neurons is mediated via the NO-cGK-

CaMKII pathway. Consistent with these studies, it has been demonstrated that

induction of LTP causes prS remodeling in organotypic slice cultures via the



                                                                                3
NMDA-NO signaling pathway (Nikonenko et al., 2003). Moreover, a recent study

demonstrated enhanced neurotransmitter vesicle endocytosis in cultured

hippocampal neurons mediated by the NO-cGMP-PIP2 pathway (Micheva et al.,

2003).

Materials and methods

Cell cultures and transfection

Briefly, hippocampi from 1 to 2 day newborn mice were dissociated through

enzymatic treatment (0.25% trypsin) and subsequent trituration. The dissociated

cells were then centrifuged at 100xg for 5 min and resuspended in 100 l mouse

neuron nucleofectorTM. Cell suspension was mixed with 3 g DNA and

transferred to the amaxa certified cuvette for electroporation using the program

0-05. Cells were then immediately resuspended in MEM and plated on coverslips

previously coated with poly (D-lysine) and laminin (Arancio et al., 1995; Ninan

and Arancio, 2004).

Drug treatments.

KN-93 (Calbiochem, California), KN-92 (Calbiochem, California), KT5823

(Calbiochem, California), L-NMA (Sigma, Missouri), D-AP5 (Sigma, Missouri) and

CNQX (Sigma, Missouri) were dissolved in the bath solution. The drugs were

perfused 10 min before and during the application of tetanus.     Forskolin and

IBMX (both from Sigma, Missouri) were dissolved in DMSO (1 M) and diluted in

the bath solution.

Electrophysiology.




                                                                              4
Neurons were held under ruptured whole-cell voltage clamp throughout the

experiment; sEPSCs were measured as described earlier (Arancio et al., 1995;

Ninan and Arancio, 2004). Two silver wires, separated by ~5 mm and close to

the surface of the coverslip, were used to evoke action potentials. Actions

potentials were evoked by passing 1 ms current pulses (using Clampex 9.2,

Axon Instruments) yielding fields of ~10V/cm through the chamber. Tetanic

stimulation to produce potentiation consisted of 3 tetani of 50Hz for 2 sec at 20
                             2+
sec interval paired with 0 Mg to allow the entrance of Ca2+ through postsynaptic

(pstS) NMDA channels (Arancio et al., 1995), presumably because high

frequency stimulation of one or a few prS neurons does not produce sufficient

depolarization of the pstS cell to expel the Mg2+ ions from the NMDA channel and

allow Ca2+ influx into the pstS neurons (Bliss and Lomo, 1973; McNaughton et

al., 1978). This protocol was chosen based on previous work showing that LTP in

cultures resembles CA1-LTP in vivo or in slices in several critical ways that

indicate that Ca2+ influx through pstS NMDA receptor channels is required for its

induction (Collingridge et al., 1983; Lynch et al., 1983; Malinow and Miller, 1986;

McNaughton et al., 1978; Wigstrom et al., 1986): a) potentiation in cultures is

blocked by perfusion with the NMDA blocker, D-APV; b) potentiation in cultures is

elicited by pairing low frequency stimulation of the prS neuron with voltage clamp

depolarization of the pstS neuron to 0 mV in the presence of Mg2+; c) potentiation

in cultures by low frequency stimulation plus pstS depolarization is blocked by D-

APV; d) potentiation in culture by high frequency stimulation in 0 Mg 2+ is blocked

when the pstS electrode contains the Ca2+ chelator BAPTA (Arancio et al., 1995).



                                                                                 5
Vesicle cycling.

      One of the methods to induce exocytosis/endocytosis in the neuronal

culture is evoking action potentials by field stimulation (Murthy and Stevens,

1998; Ryan et al., 1996) (Figure 2A). Coverslips with neurons were mounted on a

plexiglass chamber on the stage of a laser scanning confocal microscope (Nikon

D-Eclipse C1). Channel series setup was used in order to avoid any signal bleed-

through between channels, when two channels were used simultaneously.

Actions potentials were evoked by passing 1 ms current pulse as described

above. Neurons were perfused with normal saline solution (119 mM NaCl, 2.5

mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES and 30 mM glucose).

Synaptic vesicles were stained with FM4-64 by perfusing the neurons with bath

solution containing 10 M FM4-64 for 45 seconds, and eliciting 50 action

potentials at 20 Hz at the beginning of dye perfusion. The perfusion solution was

then changed back to normal bath solution for 5 min to wash off the dye from the

external medium. ADVASEP-7 (1 mM, CyDex, Inc., Overland Park, KS), an

anionic cyclodextrin complexing agent was introduced for 60 sec in the washing

bath solution at 1 min of washing for enhanced removal of the dye from the

external medium. After 5 min washing, an image was taken to record the loading

of FM dye in the synaptic btns. Then the neurons were subjected to 1200 action

potentials at 20Hz to evoke repeated cycles of exocytosis, which facilitated

release of the dye trapped in the vesicles (Supplementary Figure S6). An image

was taken after destaining. NMDA receptor antagonist, D-AP5 (40 M) and non-

NMDA receptor antagonist, CNQX (20 M) were included in the bath solution to



                                                                               6
block possible recurrent excitation and induction of activity-dependent plasticity

during loading and unloading stimulation. The difference between the images

before and after destaining gave the measure of FM dye labeling of active prS

terminals. To study tetanus-induced prS plasticity changes, 3 tetani of 50 Hz for 2

sec at 20 sec intervals during brief perfusion with Mg2+ free medium were

applied. At 30 min after the tetanus, the staining and destaining procedures were

repeated. For the measurement of single vesicle fluorescence, single action

potential was applied during perfusion of FM4-64 for 10 sec. Cultures were

viewed with 40×/1.3 nA objective. The investigator was blind to the experimental

conditions. Individual fields, (30.8 X 30.8 m) were chosen randomly and

analyzed using the software Image J. Regions of interest (ROIs) were selected

over a 4x4 pixel box for single btn analysis (Ryan et al., 1996). The average

intensity over a 4x4 pixel box around the center of mass is calculated with

contribution of background area removed by subtracting the image after

destaining. Same analysis was applied to nonsynaptic axonal regions. To

analyze GluR1-GFP puncta apposing prS btns, multiple btn areas were

excluded. In order to avoid errors due to operator judgment, all images were

subjected to exactly the same acquiring and analysis conditions. The analysis

was restricted to a subset of 3–8 contiguous images in the z-axis series. Images

in each z-series were aligned and condensed with maximum transparency.

Taking images at successive time points and looking for puncta overlap was

used to check any change in focal plan or lateral movement.




                                                                                 7
Estimation of releasable fluorescence of single vesicle

We have estimated releasable fluorescence of a single vesicle based on

previous approaches (Aravanis et al., 2003; Murthy and Stevens, 1998; Ryan et

al., 1997; Slutsky et al., 2004) but using a non-parametric method (Ninan et al.,

2006). This method is based on the assumption that a single action potential

causes release of one vesicle. Synaptic vesicles were stained with FM4-64 by

perfusing the neurons with bath solution containing 10 M FM4-64 for 10 sec,

and eliciting 1 action potential (Figure 4A). The perfusion solution was then

changed back to normal bath solution for 5 min to wash off the dye from the

external medium. ADVASEP-7 (1 mM), an anionic cyclodextrin complexing agent

was introduced for 60 sec in the washing bath solution at 1 min of washing for

enhanced removal of the dye from the external medium. After 5 min washing, an

image was taken to record the loading of FM dye in the synaptic btns. Then the

neurons were subjected to 1200 action potentials at 20Hz to evoke repeated

cycles of exocytosis, which facilitated release of the dye trapped in the vesicles.

An image was taken after destaining (Figure 4B). The releasable fluorescence

was calculated on recycling btns using Image J software. A non-parametric

estimate of the releasable fluorescence of single vesicle was calculated

(6.907 0.075) using the methods of Ninan et al. (2006) from a sample of

fluorescence measurements taken from 546 btns. The methods are based on

evaluating the sample average of a periodic function at various frequencies, and

taking the estimate to be the value of the period corresponding to the maximum

sample average. Motivation for the approach is derived from the observation that,



                                                                                 8
as long as the variability in the intensity of a single active btn loaded by eliciting

one action potential and the measurement error is small relative to the average

intensity of active btns, and as long as the distributions of the intensities and of

the error are fairly symmetric, then the empirical distribution of intensities in btns

would tend to show periodic local modes at integer multiples of the average

intensity of active btns.

Legend for Supplementary Figure S1.

Tetanus-induced increase in sEPSC frequency and amplitude is via the NO-cGK-

CaMKII pathway. A. Percentage changes in sEPSC frequency in control (n=3),

tetanus (n=8), L-NMA (50 M) paired with tetanus (n=11) and KT5823 (2 M)

paired with tetanus (n=5) groups. Perfusion with L-NMA and KT5823 blocked

tetanus-induced increase in sEPSC frequency (two-way ANOVA with repeated

measures). B. Percentage changes in sEPSC amplitude in control (n=3), tetanus

(n=8), L-NMA (50 M) paired with tetanus (n=11) and KT5823 (2 M) paired with

tetanus (n=5) groups. Perfusion with L-NMA and KT5823 blocked tetanus-

induced increase in sEPSC amplitude (two-way ANOVA with repeated

measures). C. Percentage changes in sEPSC frequency in control (n=3), tetanus

(n=8), KN-93 (5 M) paired with tetanus (n=11) and KN-92 (5 M) paired with

tetanus (n=5) groups. Perfusion with KN-93 but not KN-92 blocked tetanus-

induced increase in sEPSC frequency (two-way ANOVA with repeated

measures). D. Percentage changes in sEPSC amplitude in control (n=3), tetanus

(n=8), KN-93 (5 M) paired with tetanus (n=11) and KN-92 (5 M) paired with

tetanus (n=5) groups. Perfusion with KN-93 but not KN-92 blocked tetanus-



                                                                                    9
induced increase in sEPSC frequency (two-way ANOVA with repeated

measures). E. Average changes in sEPSC frequency in control (n=3 neurons),

tetanus (n=8 neurons), L-NMA paired with tetanus (n=11 neurons), KT5823

paired with tetanus (n=5 neurons), KN-93 paired with tetanus (n=5 neurons) and

KN-92 paired with tetanus (n=5 neurons) groups. Perfusion with L-NMA, KT5823

and KN-93 but not KN-92 reversed the effect of tetanus on mean sEPSC

frequency. *P< 0.001 compared to tetanus group. The mean sEPSC frequency

was 5.6 ± 0.2Hz, 5.5 ± 0.3Hz, 5.7 ± 0.3Hz and 8.9 ± 0.4Hz, respectively for L-

NMA + tetanus, KT-5823 + tetanus, KN-93 + tetanus and KN-92 + tetanus

groups. F. Average changes in amplitude in control (n=3), tetanus (n=8), L-NMA

paired with tetanus (n=11 neurons), KT5823 paired with tetanus (n=5 neurons),

KN-93 paired with tetanus (n=5 neurons) and KN-92 paired with tetanus (n=5

neurons) groups. Perfusion of L-NMA, KT5823 and KN-93 but not KN-92

reversed the effect of tetanus on amplitude. *P<0.001 compared to tetanus

group. The mean amplitudes were 15.6 ± 0.6pA, 16.3 ± 0.5pA, 14.6 ± 0.6pA and

20.8 ± 0.9pA for L-NMA + tetanus, KT-5823 + tetanus, KN-93 + tetanus and KN-

92 + tetanus groups.

Legend for Supplementary Figure S2

A. Examples of FM4-64 staining 8 min before the tetanus and 1, 15 and 30 min

after the tetanus. Scale bar 3 m. B. Percentage increase in number of functional

prS btns at 1, 15 and 30 min after the tetanus. C. Percentage increase in

fluorescence intensity of functional prS btns at 1, 15 and 30 min after the tetanus.

Legend for Supplementary Figure S3




                                                                                 10
A. Examples of FM4-64 staining using high K+ bath solution before and after

tetanus. Application of tetanus in absence of magnesium increased number of

prS btns but not releasable fluorescence in preexisting prS btns. Scale bar 5 m.

B. Percentage increase in number of functional prS btns 30 min after the tetanus.

C. Percentage changes in releasable fluorescence of preexisting functional prS

btns 30 min after the tetanus.

Legend for Supplementary Figure S4

Tetanus occludes the occurrence of forskolin- or IBMX-induced increase in

number of FM4-64 stained VAMP2-GFP puncta and FM4-64 intensity. A.

Experimental protocol for studying the effect of tetanus, forskolin or IBMX on

number of FM4-64 stained VAMP2-GFP puncta and FM4-64 intensity. B.

Percentage changes in number of FM4-64 stained VAMP2-GFP puncta in

control, tetanus alone, tetanus+forskolin, forskolin alone, tetanus+IBMX and

IBMX alone groups. The number of FM4-64 stained VAMP2-GFP btns in

tetanus+forskolin and tetanus+IBMX groups were not significantly different from

the tetanus alone group at any time point (two-way ANOVA). Application of

forskolin or IBMX alone produced a gradual increase in number of FM4-64

stained VAMP2-GFP puncta with maximum effect at 120 min after perfusion of

forskolin or IBMX. C. Percentage changes in FM4-64 intensity in control, tetanus

alone, tetanus+forskolin, forskolin alone, tetanus+IBMX and IBMX alone groups.

The intensity of FM4-64 btns in tetanus+forskolin and tetanus+IBMX groups were

not significantly different from the tetanus alone group at any time point (two-way

ANOVA). Application of forskolin or IBMX alone produced a gradual increase in




                                                                                11
FM4-64 fluorescence intensity with maximum effect at 120 min after perfusion of

forskolin or IBMX.

Legend for Supplementary Figure S5

The ability of pre-existing prS btns to undergo plasticity depends on initial release

probability. A. Experimental protocol for staining, destaining and application of

tetanus using electrical stimulation. B. Confocal images of low (L), medium (M)

and high (H) probability btns before and after tetanus. Scale bar 2 m. C. The

probability values are presented as a box with the boundary closest to zero

indicating the 25th percentile. A line within the box marks the median, and the

boundary of the box farthest from zero indicates the 75th percentile. Error bars

above and below the box indicate the 90th and 10th percentiles. The outlying

points are excluded from the graph. Low and medium probability prS btns

undergo SP whereas high probability prS btns did not undergo increase in

releasable fluorescence after the tetanus (WMPT). The post-tetanus probability

values are identical in all the three populations suggesting a ceiling effect on

number of vesicles available for release under our experimental conditions.

Perfusion of L-NMA (D), KT5823 (E), KN-93 (F) but not KN-92 (G) blocked

tetanus-induced enhancement of release probability in low and medium

probability btns.

Legend for Supplementary figure S6

A. Examples of destaining of FM4-64 btns during 1200 action potentials at 20 Hz.

Scale bar 2 m B. Percentage decrease in FM4-64 fluorescence intensity during

destaining with 1200 action potentials at 20Hz (n=78 btns). The percentage FM4-



                                                                                  12
64 fluorescence is calculated based on the fluorescence intensity of prS btns

immediately before the application of destaining stimulus.



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Description: Cell tetanus