Supporting Information

Article Title: Infusion of growth hormone into the hippocampus induces molecular and

behavioral responses in mice

Journal Name: Experimental Brain Research

Author names: Paitoon Srimontri, Haruna Hirota, Hiroki Kanno, Toshiya Okada,

Yoshio Hirabayashi, and Keiko Kato

Corresponding author: Keiko Kato

Faculty of Life Sciences

Kyoto Sangyo University

Motoyama, Kamigamo

Kita-ku, Kyoto 603-8555, Japan

Tel: +81-757053067

Fax: +81-757053067

E-mail: kato@cc.kyoto-su.ac.jp

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Supplemental Materials and Methods

1. Elevated plus maze test - The elevated plus maze apparatus consisted of two open

arms (25 × 5 cm), two closed arms (25 × 5 × 15 cm), and a central platform (5 × 5 cm).

The apparatus was elevated 50 cm above the floor level, and grip on the open arms was

facilitated by inclusion of a small edge (0.5-cm high) around their perimeter. The

apparatus was made of plastic materials ( O’hara and Co., Ltd., Tokyo, Japan ) , and a

fluorescent lamp was set to 350 lux, while a trial was recorded with a camera mounted

130 cm above the field floor and automatically analyzed with Time OFCR4 (O’hara and

Co., Ltd.). The number of a mouse’s entry into the open arm served as an index of

anxiety-like behavior. The elevated plus maze test was performed on days 1, 5, and 9

after the first microinjection (Fig. 1A).

2. Dark-light box test - The dark-light box test was performed on days 3, 7, and 11 after

the first microinjection (Fig. 1A). The dark-light box consisted of a cage (22 × 44 × 25

cm) with two sections of equal size divided by a partition containing a door. One

section of the apparatus was illuminated by a fluorescent lamp (350 lux), while the other

section remained dark (2 lux). Mice were placed into the dark side of the box for 30 s

after which the door (5 × 4 cm) separating the two compartments was manually opened.

Movement of the mouse was detected and recorded using an infrared camera, and data

were collected with Time OFCR4 and analyzed with the Image J program. Latency to

enter the light chamber was determined as an index of anxiety-like behavior.

3. Canonical discriminant function formulae- Because the polynominal regression

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curves with Spearman’s correlation coefficients (two-tailed) showed that levels of Arc

and Npas4 mRNAs correlated with levels of spontaneous locomotion and the number of

corner access, respectively, we next investigated whether the correlations were

dependent on the presence of rGH and pegvisomant with canonical discriminant

function formulae among rGH-, pegvisomant-, and vehicle-groups using a combination

of Excel and KyPlot 5.0. Significances of the group judgments based on the formula

were measured by the apparent error rate (%): arbitrary units (a1), activity on one day

(a2), and activity on the other day (a3).

Spontaneous locomotion and Arc in Fig 4A:

rGH group = (-5.99 - 3.90a1 - 9.49a2 + 27.51a3-9.76)2 + (-1.05 + 4.70a1 - 2.33a2 -

2.52a3 + 0.32)2,

Pegvisomant group = (9.76 - 3.90a1 - 9.49a2 + 27.51a3 - 9.76)2 + (-0.17 + 4.70a1 -

2.33a2 - 2.52a3 + 0.32)2,

Vehicle group = (-3.77 - 3.90a1 - 9.49a2 + 27.51a3 - 9.76)2 + (1.22 + 4.70a1 - 2.33a2

- 2.52a3 + 0.32)2; Canonical correlation coefficients, 0.99 (X), 0.73 (Y); Bartlett’s Chi-

square test, 2 = 39.64 (X), 6.18 (Y); Df = 6 (X), 2 (Y); ****p < 0.0001 (X), *p < 0.05 (Y);

apparent error rate, 0%.

The number of corner access and Npas4 in Fig. 4B:

rGH group =

(-0.37 + 0.57*a1 + 0.03*a2 - 0.02*a3 - 2.58)2 + (-0.13 + 0.30*a1 - 0.07*a2 + 0.07*a3 -

1.65)2

Pegvisomant group =

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(1.21 + 0.57*a1 + 0.03*a2 - 0.02*a3 - 2.58)2 + (0.03 + 0.30*a1 - 0.07*a2 + 0.07*a3 -

1.65)2

Vehicle group =

(-0.84 + 0.57*a1 + 0.03*a2 - 0.02*a3 - 2.58)2 + (0.10 + 0.30*a1 - 0.07*a2 + 0.07*a3 -

1.65)2

Canonical correlation coefficients, 0.71 (X), 0.11 (Y); Bartlett’s Chi-square test, 2 =

5.74 (X), 0.10 (Y); Df = 6 (X), 2 (Y); p = 0.45 (X), p = 0.95 (Y); apparent error rate,

16.67%.

4. Immunofluorescence - The following antibodies were used for immunofluorescence

experiments: goat anti-GHR (R&D, AF1360), mouse anti-Arc (C-7) monoclonal

antibody (Santa Cruz, sc-17839), rabbit anti-c-Fos (Abcam, ChIP ab7963), rabbit anti-

Nr4a1 (Enogene, Ab-351), rabbit anti-Npas4 (Novus, NBP1-06574), chicken anti-goat

IgG conjugated with Alexa Fluor 488 (Life technologies, A-21467), goat anti-mouse

IgG conjugated with Alexa Fluor 568 (Life technologies, A11019), and donkey anti-

rabbit IgG conjugated with Alexa Fluor 568 (Invitrogen, A10042). Following drug

microinjection and behavioral experiments, mouse brains were extracted and quickly

frozen using dry ice. Coronal brain sections (14 m) were obtained using a microtome

cryostat (Cryo-Star HM 560V, Microm International GmbH, Micro edge Instruments

Co. Ltd., Tokyo, Japan). Brain specimens were then fixed with 4% paraformaldehyde

in Dulbecco’s phosphate buffered saline at 4 °C for 30 min, washed with 0.15 M NaCl,

20 mM boric acid, and 5 mM sodium tetraborate decahydrate, pH 8.0 (BBS), and

incubated with 20 mM glycine, BBS for 10 min at room temperature. Specimens were

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then treated with 3% bovine serum albumin and 3% goat serum in BBS for 30 min at

4 °C and incubated in primary antibody in 3% bovine serum albumin, 3% goat serum,

and BBS for 16 h. After washing with BBS, sections were incubated in secondary

antibody in 3% bovine serum albumin, 3% goat serum, and BBS at 4 °C for 16 h. All

sections were treated with DAPI ( Life technologies , D21490), mounted onto slides in

Permafluor (TA-030-FM, Thermo Scientific Japan, Yokohama, Japan), and

coverslipped. Slides were then observed using an upright fluorescence microscope

(ECLIPSE 80i, Nikon Corporation, Tokyo, Japan) and pictures were obtained using a

camera (DS-Qi1Mc, Nikon) and imaging software (NIS Elements ver 4.10, Nikon).

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Supplementary Figures

Supplementary figure 1. Effects of kindled-seizures on the expression levels of Arc,

Nr4a1, Npas4, and Fos. Kindled mice were prepared as described previously (Kato et

al. 2001) and criteria of seizures were monitored with a modified-classification of

Racine's criteria (Kato et al 2001; Racine 1972). The present qRT-PCR using the

primers described in Materials and Methods amplified single bands originating from

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transcripts with a SYBR green filter in 1.4% agarose-TAE (A): S, sample with total

RNA in the caudal cortex in a mouse; DW, distilled water instead of total RNA solution.

Total RNA was isolated from the caudal cortex containing the hippocampus and

amygdaloid complex during epileptogenesis (stage 3) and following kindled-seizures

(kindling), and qRT-PCR was performed (B-D). Bar graph (Mean + S.E.M.) shows the

ratio of Arc, Npas4, and Fos mRNA relative to GAPDH mRNA between sham-operated

(n = 5), stage 3 (n = 4), and full-kindled (n = 5) mice using a one-way factorial ANOVA

(Fisher’s PLSD test): F (2, 11) = 8.77, **p < 0.01, (kindling/ sham and kindling/ stage 3,

*p < 0.01) in Arc (B); F(2, 11) = 5.34, *p < 0.05 (kindling/ sham and kindling/ stage 3, *p

< 0.05) in Nr4a1 (C); F (2, 11) = 30.34, ***p < 0.0001 (kindling/ sham and kindling/

stage 3, ****p < 0.0001) in Npas4 (C); F (2, 11) = 12.81, **p < 0.005, (kindling/ sham and

kindling/ stage 3, **p < 0.005) in Fos (D). The average levels of the transcripts of Arc,

Nr4a1, Npas4, and Fos increased to 2.4-fold, 2.1-fold, 6.5-fold, and 4.3-fold following

kindled seizures, comparing with sham-operated mice.

Additional reference:

Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor

seizure. Electroencephalogr Clin Neurophysiol 32:281-294.

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Supplementary figure 2. Effect of drugs on results of the elevated plus maze test.

The elevated plus maze test was performed on days 1, 5 and 11 following

microinjections of vehicle (n = 9), rGH (n = 8), and pegvisomant (n = 8) as shown in

Fig. 1A. The bar graph shows means + SEM of number of open arm entries (A), number

of closed arm entries (B), and total time spent in the center (C) of the plus maze field for

10 min. Effect significance for drugs was analyzed with a two-way ANOVA (Tukey’s

and Fisher’s LSD multiple comparison test): F(2,62) = 6.218, ** p < 0.005 ( ** p < 0.005

[rGH/pegvisomant] in Tukey, *p < 0.05 [rGH/vehicle] in Fisher’s LSD) in A; F(2,62) =

0.120, p = 0.89 in B; F(2,62) = 0.286, p = 0.75 in C. rGH enhanced access to the open

arm by 2.1- and 1.4-fold compared to pegvisomant and vehicle on day 1 after the first

injection, respectively.

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Supplementary figure 3. Effect of drugs on results of the dark-light box test. The

dark-light box test was performed on days 2, 6 and 10 following microinjections of

vehicle (n = 9), rGH (n = 8), and pegvisomant (n = 8) as shown in Fig. 1A. The bar

graph shows means + SEM of time (s) in the light box (A), time (s) in the dark box (B),

and latency to the light box (C) in the plus maze field for 10 min. Effect significance for

drugs was analyzed with a two-way ANOVA (Tukey’s and Fisher’s LSD multiple

comparison test): F (2, 63) = 0.621, p = 0.54 (A and B); F (2, 63) = 0.969, p = 0.38 (C).

We found no difference in the results of dark-light box tests among groups of mice that

received injections of drugs.

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Supplementary figure 4. Polynominal regression analyses between gene expression

and spontaneous locomotion in the dispersion diagram. Polynominal regression

curves of locomotion with expression of Arc (A), Nr4a1 (B), Npas4 (C), and Fos (D)

are shown. The graph with Arc (A) is the same as that shown in Fig. 4A. Polynominal

regression formulae (Spearman correlation (two-tailed), number, p-value): Nr4a1, Y =

0.472 + 0.712*X - 0.292*X2 (r = 0.38, n = 12, p = 0.22) on Day 8; Y = 0.342 + 0.638*X

+ 0.271*X2 (r = 0.69, n = 12, *p < 0.05) on Day 10 (B); Npas4, Y = 0.446 + 0.146*X -

0.013X2 (r = 0.25, n = 12, p = 0.43) on Day 8; Y = 0.111 + 0.336*X - 0.033*X2 (r =

0.40, n = 12, p = 0.19) on Day 10 (C); Fos, Y = 0.498 + 0.505*X - 0.264*X2, (r = -0.01,

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n = 12, p = 0.93) on Day 8; Y = 0.314 + 0.425*X, (r = 0.34, n = 12, p = 0.28) on Day 10

(D). Spearman correlations suggested a correlation between levels of Nr4a1 mRNA and

locomotion on Day 10.

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Supplementary figure 5. Polynominal regression analyses between gene expression

and self-grooming behavior in the dispersion diagram. Polynominal regression

curves of self-grooming with expression of Arc (A), Nr4a1 (B), Npas4 (C), and Fos (D)

are shown. Polynominal regression formulae (Spearman correlation (two-tailed),

number, p-value): Arc, Y = 32.28 + 47.63*X - 29.49*X2 (r = 0.02, n = 12, p = 0.96) on

Day 5; Y = 21.24 + 55.76*X - 49.59*X2 (r = -0.28, n = 12, p = 0.38) on Day 9 (A);

Nr4a1, Y = 15.27 + 150.3*X - 136.3*X2 (r = 0.09, n = 12, p = 0.78) on Day 5; Y =

16.48 + 105.1*X - 133.2*X2, (r = -0.09, n = 12, p = 0.78) on Day 9 (B); Npas4, Y =

16.54 + 18.27*X - 1.94*X2 (r = 0.29, n = 12, p = 0.36) on Day 5; Y =

20.90+1.55*X+0.14*X2 (r = 0.09, n = 12, p = 0.78) on Day 9 (C); Fos, Y = 102.7 -

134.3*X + 69.13*X2 (r = -0.16, n = 12, p = 0.60) on Day 5; Y = 101.9 - 146.1*X +

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61.97*X2 (r = 0.028, n = 12, p = 0.94) on Day 11 (D). Red square shows outliers in A.

Spearman correlations suggested no correlations in all present curves.

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Supplementary figure 6.　Polynominal regression analyses between gene

expression and the number of corner accesses in the dispersion diagram.

Polynominal regression curves of corner access with expression of Arc (A), Nr4a1 (B),

Npas4 (C), and Fos (D) are shown. The graph of Npas4 (C) is the same as that shown

in Fig. 4B. Polynominal regression formulae (Spearman correlation (two-tailed),

number, p-value): Arc, Y = 74.03 + 9.191*X - 11.63*X2 (r = -0.014, n = 12, p = 0.96) on

Day 5; Y = 89.80 - 31.20*X + 15.04*X2 (r = 0.12, n = 12, p = 0.72) on Day 9 (A);

Nr4a1, Y = 49.73 + 135.5*X - 138.8*X2 (r = 0.14, n = 12, p = 0.67) on Day 5; Y =

64.64 + 77.67*X - 78.05*X2 (r = 0.23, n = 12, p = 0.47) on Day 9 (B); Fos, Y = -2.75 +

226.1*X - 138.6*X2 (r = -0.16, n = 12, p = 0.60) on Day 5; Y = -21.74 + 285.5*x -

170.2*X2 (r = 0.03, n = 12, p = 0.94) on Day 9 (D). Spearman correlations suggested no

correlations in curves including Arc, Nr4a1, or Fos mRNAs.

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Supplementary figure 7. Effect of octreotide on spontaneous locomotor activity.

The bar graph shows means + SEM of locomotion as shown in Fig. 2. The mean +

SEM of locomotor counts per min for 48 h before injections of octreotide (n = 8; 90

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pmol/L; Novartis; Tokyo) were 21.0 + 1.0 in A (24 h); 17.0 + 1.1 in B (light); 25.1 +

1.4 in C (dark), respectively. Significance for effects between vehicle and octreotide

groups was analyzed with a two-way ANOVA: F (1, 30) = 0.13, p = 0.73 in A; F (1, 30)

= 9.17, **p < 0.01 in B; F (1, 30) 1.18, p = 0.29 in C.

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Supplementary figure 8. Distribution of end products of growth hormone receptor

(GHR) and Arc expression in the hippocampus following administration of drugs.

Double-labeled immunofluorescence micrograph with anti-GHR (green) and anti - Arc

(red) antibodies in the hippocampus on Day 11 following microinjections of rGH (A-H),

vehicle (I-L), and pegvisomant (M-P), which were counterstained with DAPI in the

CA3 field of the hippocampus (pcl, pyramidal cell layer; sl, stratum lucidum).

Immunofluorescence with DAPI, anti-GHR antibody, and anti- Arc antibody (A, E, I,

and M [Bar = 100 m]; D, H, L, and P [Bar = 20 m]); anti-GHR antibody (B, F, J, and

N); and anti- Arc antibody (C, G, K, and O). Representative double-positive cell (red

arrowhead) and only GHR-positive cell (red arrow) are shown in the inset (Bar =

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10 m) in D. GHR expressed in hippocampal pyramidal neurons exhibited little

variability in distribution and intensity regardless of drug administration. Several GHR-

positive cells co-expressed Arc protein in nonpyramidal neurons on the ipsilateral side

of the stratum lucidum following injection of rGH (arrowheads, A-D). Arrows show

cells expressing only GHR (A, B, D). There was little presence of double-labeled anti-

GHR and anti- Arc cells in the hippocampus of vehicle- and pegvisomant-injected

groups (L).

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Supplementary figure 9. Distribution of end products of growth hormone receptor (GHR), and Fos , Nr4a1 , and Npas4

expression in the hippocampus following administration of drugs. Double-labeled immunofluorescence micrograph with anti-GHR

(green), and anti - Fos (red) in A-F (Bar = 100 m), anti- Nr4a1 (red) in G-L, (Bar = 50 m), or anti- Npas4 (red) antibodies in M-R (Bar

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= 50 m), in the hippocampus on Day 11 following microinjections of rGH (A, B, G, H, M, and N), vehicle (C, D, I, J, O, and P), and

pegvisomant (E, F, K, L, Q, and R), which were counterstained with DAPI. Asterisks indicate dust (G, H, and Q). Staining with DAPI,

anti-GHR antibody, and anti- Fos antibody (A, C, and E); DAPI, anti-GHR antibody, and anti- Nr4a1 antibody (G, I, and K); DAPI, anti-

GHR antibody, and anti- Npas4 antibody (M, O, and Q); anti- F os (B, D, and F); anti- Nr4a1 antibody in (H, J, and L); and anti- Npas4

antibody (N, P, and R). Abbreviations: GL, granular cell layer; ML, molecular layer; PoGL, polymorphic layer of the dentate gyrus; pcl,

pyramidal cell layer; sl, stratum lucidum of the CA3-field of the hippocampus. Fos was expressed in the nuclei of granule cells of the

dentate gyrus; there was little difference in the expression of Fos among rGH-, vehicle-, and pegvisomant-treated groups (A-F). The

number of GHR-positive cells in the polymorphic layer of the dentate gyrus also increased following injection of rGH (A). Nr4a1 and

Npas4 proteins expressed in nuclei of hippocampal pyramidal cells (H, J, L, N, P, R). There was little difference in signal intensity of

Nr4a1 protein among rGH-, vehicle-, and pegvisomant-groups, however, signal intensity of Npas4 protein decreased in pegvisomant-

group (R). Additionally, there was no presence of double-labeled cells with anti-GHR and anti- Fos ; however, a pyramidal cell was

double-labeled with anti-GHR and the other antibodies including anti- Nr4a1 , and anti- Npas4 in the hippocampus.

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Table S1. Expression ratio of immediate early genes (IEGs) in the brain following

kindled seizures relative to sham-operated mice

Go biological

process/

molecular

function/ cellular

component

terms

Gene

symbolGene name Primer set ID

Fold change

Apical

cortex

*

Caudal

cortex

Transcription

Npas4Neuronal PAS domain

protein 41459372_at 11.3 16.0

Fos FBJ osteosarcoma oncogene 1423100_at 8.0 11.3

Dkk1 Dickkopf homolog 1 1458232_at 1## 10.6

Nfkbiz

Nuclear factor of kappa light

polypeptide gene enhancer in

B-cells inhibitor, zeta

1448728_a_at 8.0 1

Egr2# Early growth response 2 1427682_a_at 6.5 5.7

Nr4a1Nuclear receptor subfamily

4, group A, member 11416505_at 3.2 3.5

Junb# Jun-B oncogene 1415899_at 3.0 4.0

Btg2#B-cell translocation gene 2,

anti-proliferative1416250_at 4.3 7.5

Fosb# FBJ osteosarcoma oncogene B 1422134_at 2.8 4.6

Zfp36 Zinc finger protein 36 1452519_a_at 2.8 3.2

Arc

Activity regulated

cytoskeletal-associated

protein1418687_at 3.7 4.6

Cytoskeleton S100a9S100 calcium binding protein

A91448756_at 11.3 1

n.r., not recognized. ##1, no difference between kindled- and sham-operated mice.

*Affimetrix gene chip array (Mouse Genome 430 2.0 array) was performed using the

mRNA extracted from the apical part of the cerebral cortex and caudal part of the

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cerebral cortex, which includes the posterior hippocampus, amygdaloid complex, and

temporal lobe. Among the genes showing >3-fold expression level following kindled

seizures, the genes of transcription- and cytoskeleton-related molecules are shown in

this table.

#There were few differences in Egr2 and Btg2 mRNAs following kindled seizures, as

determined using qRT-PCR (data not shown). Increases of Junb and Fosb mRNAs were

less than two times, as determined using qRT-PCR (Kato et al. 2009).

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