GABA from reactive astrocytes impairs memory in mouse … · 2014-08-06 · Gat1 in astrocytes at3 i n ap + U) 40 20 0 60 WT APP/PS1 10 9 Gat3 in astrocytes NS Gfap / Gad67 / GABA

SUPPLEMENTARY INFORMATION

GABA from reactive astrocytes impairs memory in mouse

models of Alzheimer disease

Seonmi Jo

*, Oleg Yarishkin

*, Yu Jin Hwang, Ye Eun Chun, Mijeong Park, Dong Ho Woo, Jin

Young Bae, Taekeun Kim, Jaekwang Lee, Heejung Chun, Hyun Jung Park, Da Yong Lee,

Jinpyo Hong, Hye Yun Kim, Soo-Jin Oh, Seung Ju Park, Hyo Lee, Bo-Eun Yoon, YoungSoo

Kim, Yong Jeong, Insop Shim, Yong Chul Bae, Jeiwon Cho, Neil W. Kowall, Hoon Ryu,

Eunmi Hwang, Daesoo Kim† and C. Justin Lee

†

Nature Medicine doi:10.1038/nm.3639

Nu

mber

of p

laqu

es

0

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ba2 months 4 months 6 months

8 months 11 months 23 months

300 mm

Plaque

WT APP/PS1

2 4 6 8 11 23All

Age (month)

c

Wild

-typ

eA

PP

/PS

1

GfapPlaque Iba1 Merge + DAPI

Wild

-typ

eA

PP

/PS

1

GfapPlaque Iba1 Merge + DAPI

200 mm

50 mm

Plaque / Gfap / Iba1 / DAPI

d

SFig 1

eDG CA1 CA3

Co

ntr

ol

Ka

inic

acid

CA3

Wild

-typ

eA

PP

/PS

1

PI

DAPI

50 mm 10 mm


Supplementary Figure 1. Amyloid plaques, reactive astrocytes, and a lack of neuronal

death.

(a) Thioflavin-S staining of amyloid plaques in the hippocampus. APP/PS1 mice start to

develop amyloid plaques at the age of 4 months and develop more with aging.

(b) Average number of plaques as a function of age (a). The number sigmoidally increases

with age while wild-type mice show no plaque at any ages tested. WT: wild-type. 6 slices

were analyzed for each group. Data are means SEM (error bars).

(c) Confocal images show numerous reactive astrocytes and activated microglia clustered

around amyloid plaques in APP/PS1 mice at 9 months of age. Gfap: glial fibrillary acidic

protein, Iba1: ionized calcium binding adaptor molecule-1. DAPI: 4', 6-diamidino-2-

phenylindole.

(d) High-magnification images of the molecular layer of DG from white box in (c).

(e) Confocal images of propidium iodide (PI) and DAPI staining show no apparent cell death

in the hippocampus of both wild-type and APP/PS1 mice (11–12 months of age). For positive

control, wild-type mice were injected with kainic acid (25 mg kg-1

, i.p.) 48 h before PI

injection. CA: Ammon’s horn of hippocampus (Cornu Ammonis), DG: dentate gyrus.


Wild-type 5XFAD

Gfap

GABA

Merge

30 mm

MoDG

GrDG

Pla

qu

e/

Gfa

p/

GA

BA

/ D

AP

I

2 months

Wild-type APP/PS1

4 months 23 months8 months6 months 11 months2 months 23 months

20 mm

Gfap

Plaque

GABA

Merge + DAPI

20 mm

Gfap

Plaque

GABA

Merge + DAPI

a b

d

SFig 2

c APP/PS1

GF

AP

/ G

AB

A

Gfa

p/

Iba

1/ G

AB

A

Gfap

Iba1

GABA

Gfap

GABA

Iba1

GABA

20 mm

Supplementary Figure 2. GABA in the reactive astrocytes of APP/PS1 and 5XFAD mice.

(a) Representative confocal images of thioflavin-S staining, Gfap and GABA

immunostaining in the molecular layer of DG. Wild-type mice didn’t show amyloid plaques


and GABA-containing reactive astrocytes even at the age of 23 months. Gfap-negative and

GABA-positive staining indicates GABAergic interneurons.

(b) Representative confocal images of APP/PS1 mice at various ages. APP/PS1 mice start to

develop amyloid plaques at the age of 4 months. Although the exact level of Gfap and GABA

varies among amyloid plaques, GABA-positive reactive astrocytes are consistently observed

at all ages with amyloid plaques.

(c) Representative confocal images of Gfap, Iba1, and GABA immunostaining in the

molecular layer of DG. (12 months of age) show that GABA is not colocalized with Iba1 but

co-localized with Gfap.

(d) Representative confocal images of 14-month-old 5XFAD mice. GABA is accumulated in

reactive astrocytes of 5XFAD mice.


a bW

ild-t

ype

AP

P/P

S1

Gfap Gad67 GABA Merge

10 mm

Wild

-type

AP

P/P

S1

Gfap Gad65 GABA Merge

10 mm

c d

e f g hGad67 in astrocytes

Inte

nsity o

f G

ad6

7

in G

fap

+p

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AU

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NS

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ad6

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aoa

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+p

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5 5

NS

Gad65 in astrocytes Maoa in astrocytes Abat in astrocytes

Inte

nsity o

f A

bat

in G

fap

+p

ixe

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AU

)

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80

0

240

18 13

NS

Wild

-type

AP

P/P

S1

Gfap Maoa GABA Merge

10 mm

Wild

-type

AP

P/P

S1

Gfap Abat GABA Merge

10 mm

i

Wild

-typ

eA

PP

/PS

1

Gfap GABAGat1 Merge + DAPI

Gfap / Gat1 / GABA / DAPI

20 mm

j

Wild

-typ

eA

PP

/PS

1

Gfap GABAGat3

Gfap / Gat3 / GABA / DAPI

Merge + DAPI

20 mm

Inte

nsity o

f G

at1

inG

fap

+p

ixe

ls (

AU

)

40

20

0

60

WT APP/PS1

10 8

NS

Gat1 in astrocytes

Inte

nsity o

f G

at3

inG

fap

+p

ixe

ls (

AU

)

40

20

0

60

WT APP/PS1

10 9

Gat3 in astrocytes

NS

Gfap / Gad67 / GABA Gfap / Gad65 / GABA

Gfap / Maoa / GABA Gfap / Abat / GABA


Supplementary Figure 3. Gad67, Gad65, Maoa, Abat, Gat1, and Gat3 expression in

reactive astrocytes.

(a–d) Expression of Gad67 (a), Gad65 (b), Maoa (c), and Abat (d) in the molecular layer of

DG was tested by immunostaining. Each enzyme is minimally expressed in astrocytes and

not changed in GABA-containing reactive astrocytes near the amyloid plaque (9 months of

age).

(e–h) Quantification of confocal images. Mean intensity of Gad67 (e), Gad65 (f), Maoa (g),

and Abat (h) was measured in Gfap-positive pixels. NS

P > 0.05 (t-test).

(i–j) Expression of Gat1 (i) and Gat3 (j) in the molecular layer of DG was tested by

immunostaining. Mean intensity of Gat1 (i) or Gat3 (j) was measured in Gfap-positive pixels.

Expression of both transporters is not changed in GABA-containing reactive astrocytes near

the amyloid plaque (9 months of age). NS

P > 0.05 (t-test).

Gad: glutamate decarboxylase. Maoa: monoamine oxidase A, Abat: GABA transaminase,

Gat: GABA transporter, WT: wild-type. AU: arbitrary unit. Number on each bar refers to the

number of cells analyzed. Data are means ± SEM (error bars).


eN-acetyl-γ-

aminobutyratePutrescine

N-acetyl-γ-

aminobutyraldehyde

Mono-acetyl

putrescine

MAOBSAT UnknownGABA

ALDH2

Acetyl-CoA Coenzyme A, H+ O2, H2O H2O2, NH4+ NAD+, H2O 2H+, NADH H2O Acetate

Wild-type APP/PS1

Gfap

Maob

Merge

50 mm

gf Wild-type APP/PS1

Gfap

Merge

GABA

Maob

10 mm

a

Wild

-type

AP

P/P

S1

Putrescine Merge + DAPIGfap GABA

Gfap / Putrescine / GABA / DAPI

MoDG

GrDG

b

Wild

-typ

e(G

fap-G

FP

)A

PP

/PS

1A

PP

/PS

1

20 mmGfap-GFP Putrescine Merge

Gfap Putrescine Merge

Iba1 Putrescine Merge

h i

Ma

ob

activity (

mo

lg

-1h

r-1)

0.4

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0

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0.8

Water Sele

4 4

***

Ma

oa

activity (

mo

lg

-1h

r-1)

0.2

0.1

0

0.3

Water Sele

4 4

NS

j

Maob

Maoa

Water Selegiline

SFig 4G

fap

/ M

aob

Gfa

p/ M

aob

/ G

AB

A

Putrescine

GF

PG

fap

Iba

1

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in G

fap

+pix

els

(A

U)

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c**

10 10

WT APP/PS1

50

dP

ears

on’s

corr

ela

tion

0.2

0.4

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Gfap-GFP

6

Gfap Iba1

WT APP/PS1

0.1

0.3

50 mm

Merge


Supplementary Figure 4. Putrescine, Maob expression, and Maob enzyme activity.

(a) Presence of putrescine, a GABA precursor, was detected by immunostaining.

Representative confocal images show global existence of putrescine in the DG molecular

layer including Gfap-positive areas. Putrescine is increased in APP/PS1 mice, and most

intense staining is observed around amyloid plaques (arrowheads) and near the GABA-

containing reactive astrocytes. In both genotypes, putrescine is absent in neurons which are

clustered at the granule cell layer (10 months of age).

(b) Confocal images show that intense staining of putrescine is well colocalized with Iba1-

positive microglia in APP/PS1 mice (10 months of age).

(c) Quantification of confocal images. Mean intensity of putrescine was measured in Gfap-

positive pixels. **

P < 0.01 (t-test).

(d) Pearson’s correlation coefficients show that putrescine is colocalized with Iba1 better than

Gfap or Gfap-GFP.

(e) Putrescine degradation III pathway for GABA production. MAOB mediates the second

reaction step. SAT1: putrescine acetyltransferase, ALDH2: mitochondrial aldehyde

dehydrogenase.

(f–g) Confocal images representing labeling with two commercially available antibodies,

which are directed against different epitopes, show increased expression of Maob in GABA-

containing reactive astrocytes of APP/PS1 mice (10 months of age). Antibodies were

purchased from Santa Cruz Inc.(f) and Sigma Inc.(g).

(h) Colorimetric enzymatic activity assay of Maob. Maob activity in the hippocampus is

selectively inhibited by drinking water administration of selegiline (5–10 mg kg-1

day-1

for 3

days) in wild-type mice (13 months of age).

(i) Maob activity after selegiline administration. Sele: selegiline. ***

P < 0.001 (t-test).

(j) Maoa activity after selegiline administration. n.s.

P > 0.05 (t-test).

AU: arbitrary unit. Number on each bar refers to the number of hippocampi analyzed. Data

are means ± SEM (error bars).


0.4

1.2

1.6

0

Ab

monom

er

norm

aliz

ed t

o b

-actin

Water Sele

APP/PS1

3 3

NS

b c

e f

5 pA

500 ms

Wild-type

APP/PS1

0

0.2

0.4

0.6

0.8

1.0

0

sEPSC peak amplitude (pA)

Cum

ula

tive p

robabili

ty

10 20 30

Wild-typeAPP/PS1

Am

plit

ude (

pA

)

6

3

011 11

**

5 15 250

0.2

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sEPSC interevent interval (ms)

Cum

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tive p

robabili

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2,000 4,000 6,000

Wild-typeAPP/PS1

Fre

quency (

Hz)

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011 11

*

a

d

5 pA

500 ms

Wild-type + TTX

APP/PS1 + TTX

0

0.2

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mEPSC peak amplitude (pA)

Cum

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robabili

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Wild-type + TTXAPP/PS1 + TTX

Am

plit

ude (

pA

)

6

3

010 10

*

5 150

0.2

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mEPSC interevent interval (ms)

Cum

ula

tive p

robabili

ty

2,000 4,000 6,000

Wild-type + TTXAPP/PS1 + TTX

Fre

quency (

Hz)

1.5

0.5

010 10

NS

1.0

20 25

mEPSC

sEPSC

SFig 5

g

PP

Stim

Patch-+

100 pA

50 ms

Selegiline

Wild-type APP/PS1

Control SelegilineControl

● Wild-type + control ● Wild-type + selegiline● APP/PS1 + control● APP/PS1 + selegiline

Stimulus intensity (mA)0 100 200 300 400 500

eE

PS

Cam

plit

ude (

pA

)

200

400

600

800

0 eE

PS

Ca

mp

litu

de

(pA

)200

400

600

800

0Cont Sele Cont Sele

Wild-type APP/PS1

9 109 10

**NS

NS

h i

j

hAPP

TetramerTrimerDimer

Monomer

b-actin

Water Selegiline

12 12 10.5 12 12 10.5 Age

(month)

0.2

0.4

0.8

0

Ab

olig

om

er

norm

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ed t

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-actin

Water Sele

APP/PS1

3 3

NS

0.6

0.8

Supplementary Figure 5. Spontaneous EPSCs (sEPSC), miniature EPSCs (mEPSC),

input-output relationship, and Ab species in DG of wild-type and APP/PS1 mice.

(a–c) sEPSCs recorded from granule cells of DG in wild-type and APP/PS1 mice (N = 2 for

each group; both sexes at 12–12.5 months of age).

(a) Representative traces of sEPSCs.

(b) Cumulative probability of sEPSC peak amplitude. Inset bar graph: Average amplitude of

sEPSCs. **

P < 0.01 (t-test).

(c) Cumulative probability of sEPSC interevent interval. Inset bar graph: Average frequency

of sEPSCs. * P < 0.05 (t-test).


(d–f) mEPSCs recorded from granule cells of DG in wild-type and APP/PS1 mice (N = 2 for

each group; both sexes at 12–12.5 months of age).

(d) Representative traces of mEPSCs.

(e) Cumulative probability of mEPSC peak amplitude. Inset bar graph: Average amplitude of

mEPSCs.*P < 0.05 (t-test).

(f) Cumulative probability of mEPSC interevent interval. Inset bar graph: Average frequency

of mEPSCs. NS

P > 0.05 (t-test).

(g–i) Input-output relationship of granule cells of DG in wild-type and APP/PS1 mice with or

without pretreatment of selegiline (100 mM) (N = 2 for each group; both sexes at 12–12.5

months of age).

(g) Schematic diagram for the input-output relationship experiments and representative traces

of eEPSCs evoked by stimuli of 300 mA intensity.

(h) Relationships between stimulus intensity and eEPSC amplitude. **

P < 0.01 for genotype, NS

P > 0.05 for drug (Two-way repeated measures ANOVA). ***

P < 0.001 for APP/PS1 +

selegiline at 50 ms (One-way ANOVA and bonferroni test).

(i) Mean amplitudes of eEPSCs evoked by stimuli of 300 mA intensity. **

P < 0.01 for

genotype, NS

P > 0.05 for drug (Two-way ANOVA).

(j) Effect of selegiline on the level of Ab oligomer and monomer in APP/PS1 mice. Western

blots of Ab in DG using 6E10 antibody (N = 3 for each group; male at 10.5–12 months of

age) revealed that the level of Ab monomer and oligomer are not significantly altered by oral

administration of selegiline for 1 week. NS

P > 0.05 (t-test).

EPSC: excitatory post-synaptic current. Number on each bar refers to the number of cells (b,

c, e, f, i) and mice (j) analyzed. N refers to the number of animals studied. Data are means (c,

f, h, i, j) or medians (b, e) ± SEM (error bars).


f

Control Selegiline

APP/PS1

e APP/PS1

Sp

ike

pro

ba

bili

ty

0.6

0.4

1.0

0.8

0.2

0

0 400 600 800 1,000200

● Control (n = 5)

● Selegiline (n = 3)

50 mV

50 ms

Stimulus intensity (mA)

c d

a

Before + BIC, CGP Before + BIC, CGP

Wild-type

Wild-type + selegilineWild-type + water

Probabilitymax

ES50

Spik

e p

rob

abili

ty


Sp

ike

pro

ba

bili

ty

0.6

0.4

1.0

0.8

0.2

0

0 400 600 800 1,000200


0.6

0.4

1.0

0.8

0.2

0

0 400 600 800 1,000200

● Before (n = 15)

● + BIC, CGP (n = 15)

● Before (n = 12)

● + BIC, CGP (n = 12)

bWild-type

SFig 6

50 mV

50 ms

50 mV

50 ms

Supplementary Figure 6. Effect of selegiline on spike probability of dentate granule cells

in wild-type and APP/PS1 mice.

(a–d) Selegiline has no apparent effect on spike probability in wild-type littermates (N = 2 for

WT + water; N = 3 for WT + selegiline; both sexes at 12–13 months of age).

(a) Example traces of evoked EPSP and action potential in wild-type mice at 300 mA

stimulation, before and after bath application of GABA receptor antagonists (BIC: 10 mM

bicuculline. CGP: 5 mM CGP55845).

(b) Example traces in selegiline-administrated wild-type mice (5–10 mg kg-1

day-1

, oral

administration for 7 days) at 300 mA stimulation, before and after bath application of GABA

receptor antagonists.

(c) Summary graph of spike probability in wild-type mice versus stimulus intensity.

(d) Summary graph of spike probability in selegiline-administered wild-type mice versus

stimulus intensity.


(e–f) Incubation of slices with selegiline (100 mM, 2 h) has a similar effect with its oral

administration in APP/PS1 mice (13 months of age).

(e) Example traces in APP/PS1 mice at 300 mA stimulation, incubation of slices with or

without selegiline.

(f) Summary graph of spike probability in APP/PS1 mice versus stimulus intensity.

EPSP: excitatory post-synaptic potential, Probabilitymax: maximum probability, ES50: 50%

effective stimulus. n refers to the number of cells analyzed. N refers to the number of animals

studied. Data are means ± SEM (error bars).


Day 15 Day 29Day 1

Handling

Hidden platform

Spatial cues

Releasing points

SFig 7E

sca

pe

la

ten

cy (

s)

10

20

30

40

60

1

Training day2 3 4 5 6

● Wild-type + water● Wild-type + selegiline● APP/PS1 + water● APP/PS1 + selegiline

50

7 8 9 10 11 12 13 14

Sw

im s

peed (

cm

s-1

)

0

10

20

30

● Wild-type + water● Wild-type + selegiline● APP/PS1 + water● APP/PS1 + selegiline

1

Training day2 3 4 5 6 7 8 9 10 11 12 13 14

a b

c d

e

Tim

e s

pen

t in

qua

dra

nts

(%

)

0

10

20

30

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Target

● Wild-type + water● Wild-type + selegiline● APP/PS1 + water● APP/PS1 + selegiline40

Right Left Opposite

f

Ta

rge

t cro

ssin

g n

um

ber

0

2

4

6

8 4 7 4

Water Sele Water Sele

Wild-type APP/PS1

Sw

im s

pee

d (

cm

s-1

)

0

10

20

30

8 4 7 4


Wild-type APP/PS1

g

8 4 7 4

Acquisition Acquisition

1st probe test 1st probe test 1st probe test

h

Tim

e s

pen

t in

qua

dra

nts

(%

)

0

10

20

30

50

Target

● Wild-type + water● Wild-type + selegiline● APP/PS1 + water● APP/PS1 + selegiline40

Right Left Opposite

i

Ta

rge

t cro

ssin

g n

um

ber

0

2

4

6

8 4 7 4


Wild-type APP/PS1

Sw

im s

pee

d (

cm

s-1

)

0

10

20

30

8 4 7 4


Wild-type APP/PS1

j

8 4 7 4

2nd probe test 2nd probe test 2nd probe test

* ****NS

Day 23

Supplementary Figure 7. Morris water maze experiment for testing learning and

memory in wild-type and APP/PS1 mice treated or non-treated with selegiline.

(a–b) Experimental protocol for Morris water maze test for wild-type and APP/PS1 mice

with or without oral administration of selegiline (both sexes at 10–12 months of age). After

selegiline administration (10 mg kg-1

day-1

for 14 days), mice were released in a Morris water

maze with a hidden platform and spatial cues. Acquisition is tested by 4 trials day-1

for 14

days. Probe test was done on the 23th

and 29th

day in the absence of the platform.

(c) Escape latency during acquisition test.


(d) Swimming speed during acquisition test.

(e) Time spent in each quadrant during 1st probe test.

(f) Target crossing number during 1st

probe test.

(g) Swimming speed during 1st probe test.

(h) Time spent in each quadrant during 2nd

probe test.

(i) Target crossing number during 2nd

probe test.

(j) Swimming speed during 2nd

probe test.

For escape latency, two-way repeated measures ANOVA followed by Bonferroni’s post hoc

analysis revealed significant effects for the genotype [F(1,88) = 35.977, P = 0.000], no drug

effect, and significant interaction effect between genotype and drug [F(1,88) = 9.411, P =

0.003]. **

P < 0.01, ***

P < 0.001, NS

P > 0.05. Difference in swim speeds and probe test results

among groups are not significant. Number on each bar refers to the number of mice (e–j)

analyzed. Data are means ± SEM (error bars).


b

a

Reactive astrocyte

(c) MAOB

(a) Ab plaque

(b) Putrescine

GABAAR

GABABR

Pre

(e) BEST1

Post

GABA release

(f) GABAR

(d) GABA

GABA from reactive astrocytes

Decrease in presynaptic release (PP-DG synapse)

Impairment in spike probability and synaptic plasticity

Learning and memory impairment

Cl–

Synapse

SFig 8

Supplementary Figure 8. Model diagram of memory impairment in AD.

In AD, astrocytes near Ab plaques (a) have more putrescine (b). Putrescine is degraded by

MAOB (c) to produce the inhibitory neurotransmitter GABA (d). GABA is then abnormally

released via BEST1 (e) which is redistributed away from microdomains. Then the released

GABA binds to extrasynaptic GABAA and GABAB receptors (f) and strongly inhibits

presynaptic release and spike probability. Consequently, granule cells of the dentate gyrus

receive less glutamatergic inputs at perforant path synapses and show reduced synaptic

plasticity. This finally leads to memory impairment in Alzheimer disease.

Pre: presynaptic terminal, Post: postsynapse, NMDAR: N-methyl-D-aspartate receptor,

AMPAR: -amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor.


c

d

a bContralateral Stab wound injury Contralateral Stab wound injury

30 mm

Gfap

GABA

NeuN

CA1 Py

SR

Mergee

Inte

nsity o

f

Gfa

p(A

U) 30

20

0

40

Cont Injury

6 5

*

Inte

nsity o

f G

AB

A

In G

fap

+p

ixe

ls (

AU

)

2,000

1,000

0

3,000

Cont Injury

6 5

***

Inte

nsity o

f

neu

ron

al G

AB

A (

AU

)

100

50

0

150

Cont Injury

40 25

NS

10

SFig 9

Supplementary Figure 9. GABA in the reactive astrocytes which is induced by stab

wound injury in the CA1 of hippocampus.

(a) Target site for stab wound injury. Stab wounds were stereotaxically introduced into the

hippocampus (blue arrow). CA1 region adjacent to the injured site (red box) and contralateral

non-injured site was observed (N = 3 for each group; males at 10 weeks of age)

(b) Representative confocal images for stab wound injury and the contralateral site.

(c) Mean intensity of Gfap. *P < 0.05.

(d) Mean intensity of GABA in Gfap-positive area. ***

P < 0.001.

(e) Mean intensity of interneuronal GABA. NS

P > 0.05.

AU: arbitrary unit. Number on each bar refers to the number of slices (c, d) or cells (e)

analyzed. N refers to the number of animals studied. Data are means ± SEM (error bars).


Supplementary Table 1. 50% effective stimulus (ES50), maximum spike probability

(Probabilitymax), resting membrane potential (RMP), and input resistance (Rinput) of

granule cells (n refers to the number of granule cells recorded).

GenotypeOral

administrationBath application n ES50 (mA) ± SEM Probabilitymax ± SEM

Wild-type Water 18 229.482 ± 1.470 0.989 ± 0.008

Selegiline 15 197.288 ± 0.752 0.993 ± 0.007

APP/PS1 Water 21 415.956 ± 6.651 0.638 ± 0.098

Selegiline 23 227.438 ± 4.795 0.974 ± 0.018

Wild-type Water before 8 220.853 ± 7.375 0.983 ± 0.011

+BIC,CGP 8 159.374 ± 1.625 1.000 ± 0.000

Selegiline before 15 231.155 ± 4.544 0.993 ± 0.007

+BIC,CGP 15 187.384 ± 7.309 1.000 ± 0.000

APP/PS1 Water before 15 415.093 ± 4.570 0.713 ± 0.112

+BIC,CGP 15 269.059 ± 4.171 1.000 ± 0.000

Selegiline before 11 206.270 ± 0.874 0.973 ± 0.027

+BIC,CGP 11 177.490 ± 0.008 1.000 ± 0.000

GenotypeOral

administrationBath application RMP (mV) ± SEM RInput (MW) ± SEM

Wild-type Water 29 -79.379 ± 1.130 284.724 ± 19.205

Selegiline 18 -81.500 ± 0.085 295.444 ± 32.218

APP/PS1 Water 21 -79.238 ± 1.494 315.381 ± 24.593

Selegiline 23 -80.652 ± 0.732 323.522 ± 18.745


Supplementary Table 2. Detailed information for statistical analysis of behavioral

test results.

Behavior tests Result from statistical analysis

Passive avoidance test

Two-way repeated measures ANOVA (at the 0.05 level)

Genotype effect: F(1,42) = 18.630, P = 0.000

Drug effect: F(1,42) = 12.494, P = 0.001

Genotype Drug interaction: not significant

One-way repeated measures ANOVA

F(3,42) = 14.060, P = 0.000

Scheffe's post hoc analysis:

WT + water vs. APP/PS1 + water, P = 0.000

WT + selegiline vs. APP/PS1 + water, P = 0.000

APP/PS1+water vs. APP/PS1 + selegiline, P = 0.023

One-way ANOVA with Scheffe's post hoc analysis (for each day)

Day 1 : WT + water vs. WT + selegiline, P = 0.004


WT + selegiline vs. APP/PS1 + selegiline, P = 0.043

Day 2 : WT + water vs. APP/PS1 + water, P = 0.000


APP/PS1 + water vs. APP/PS1 + selegiline, P =0.013

Morris water maze test

Two-way repeated measures ANOVA

Genotype effect: F(1,88) = 35.977, P = 0.000

Drug effect: not significant

Genotype Drug interaction: F(1,88) = 9.411, P = 0.003

One-way repeated measures ANOVA

F(3, 88) = 21.468, P = 0.000

Scheffe's post hoc analysis:

WT + water vs. APP/PS1 + water, P = 0.000

WT + water vs. APP/PS1 + selegiline, P = 0.034


APP/PS1 + water vs. APP/PS1 + selegiline, P = 0.012

One-way ANOVA with Scheffe's post hoc analysis (for each day)

Day 1–6: not significant

Day 7: WT + water vs. APP/PS1 + water, P = 0.048






Day 13: WT + water vs. APP/PS1 + water, P =0.004

Day 14: not significant


Supplementary Table 3. Human tissue information for control subjects and subjects

with Alzheimer disease.

Case Age Sex Braak stage Cause of death

Control 1 87 F I Unknown

Control 2 88 M I Adenocarcinoma

Control 3 86 M II Unknown

Control 4 87 F II Unknown

Control 5 67 M I Sudden accidental death

Control 6 82 M I Colon cancer

Control 7 61 M I Unknown

Control 8 101 F I Myocardial infarction

Control 9 89 M III Renal failure

Control 10 68 M I Congestive heart failure

Control 11 78 F I Unknown

AD 1 82 M V AD

AD 2 79 F VI AD

AD 3 70 M VI AD

AD 4 59 M VI AD

AD 5 80 F V AD

AD 6 92 M V AD

AD 7 90 F V AD

AD 8 100 M V AD

AD 9 75 M V AD

AD 10 83 M VI AD

AD 11 79 F VI AD


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