᪥᫬䠖ᖹᡂ 㻞㻤 ᖺ 㻥 ᭶ 㻞 ᪥䠄㔠䠅 ᫬ ศ㹼 ᫬ ศ㸦 㛤ሙ㸧㻌

V. 化学物質リスク研究事業・班会議資料

平成 28 年 9 月 2 日開催

ᖹᡂ㻞㻤ᖺᗘཌ⏕ປാ⛉Ꮫ◊✲㈝⿵ຓ㔠䠄໬Ꮫ≀㉁䝸䝇䜽◊✲஦ᴗ䠅㻌 䛂Ⓨ㐩ᮇ䛻䛚䛡䜛⤫ྜⓗ䛺㐜Ⓨᛶ⚄⤒ẘᛶヨ㦂ἲ䛾㛤Ⓨ䛃㻌

䠄◊✲ㄢ㢟␒ྕ䠖㻴㻞㻤㻙໬Ꮫ㻙୍⯡㻙㻜㻜㻟䠅㻌 ᖹᡂ 㻞㻤 ᖺᗘ㻌 ➨ 㻝 ᅇ⌜఍㆟㻌 ㆟஦ḟ➨㻌

᪥᫬䠖ᖹᡂ 㻞㻤 ᖺ 㻥 ᭶ 㻞 ᪥䠄㔠䠅 ᫬ ศ㹼 ᫬ ศ㸦 㛤ሙ㸧㻌

ሙᡤ䠖ඵ㔜Ὢ೜ᴦ㒊㻌 ➨ 㻣 ఍㆟ᐊ㻌

䠄䛈㻝㻜㻠㻙㻜㻜㻞㻤㻌 ᮾி㒔୰ኸ༊ඵ㔜Ὢ 㻞㻙㻝䠅㻌 㼔㼠㼠㼜㻦㻛㻛㼣㼣㼣㻚㼥㼍㼑㼏㼔㼕㼗㼍㻚㼏㼛㼙㻛㼍㼏㼏㼑㼟㼟㻚㼜㼔㼜㻌

㆟஦ḟ➨䠖㻌

䠍䠊䛿䛨䜑䛻㻌 䠄㻝㻟㻦㻟㻜䡚㻝㻠㻦㻜㻜䠅㻌 㛤఍䛾ᣵᣜ㻌

⮬ᕫ⤂௓㻌

㻞䠊ᮏ◊✲⌜䛾㐍䜑᪉䠄ㅋ⏣䠅㻌 䠄㻝㻠㻦㻜㻜䡚㻝㻠㻦㻟㻜䠅㻌

㻟䠊ྛศᢸ◊✲⪅䜘䜚ሗ࿌䠄㻝㻠㻦㻟㻜䡚㻝㻤㻦㻜㻜䠅㻌

㐃⤡஦㡯䠄㻝㻤㻦㻜㻜䡚㻝㻤㻦㻝㻡䠅

௒ᚋࡢࢫࢣࢪ࣮ࣗࣝ

㛢఍ࡢᣵᣜ

௨ୖ㻌

Ⓨ㐩ᮇ䛻䛚䛡䜛⤫ྜⓗ䛺 㐜Ⓨᛶ⚄⤒ẘᛶヨ㦂ἲ䛾㛤Ⓨ

ᖹᡂ28ᖺ9᭶2᪥

ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ

⸆⌮㒊 ㅋ⏣ Ὀᡂ

0+*5

ᖹᡂ28ᖺᗘཌ⏕ປാ⛉Ꮫ◊✲㈝⿵ຓ㔠 䠄໬Ꮫ≀㉁䝸䝇䜽◊✲஦ᴗ䠅

H28ᖺᗘ H29ᖺᗘ H30ᖺᗘ

ホ౯ᣦᶆ䛾☜❧

䝥䝻䝖䝁䝹䛾᭱㐺໬

᭷⏝ᛶ䞉ண ᛶ䛾᳨ド

ᅜ㝿ᶆ‽໬䛻ྥ䛡䛯᳨ド䠄䝥䝻䝖䝁䝹䛾ᥦ᱌䠅 H31௨㝆

H34௨㝆

䝥䝺䝞䝸䝕䞊䝅䝵䞁 䝞䝸䝕䞊䝅䝵䞁

HESI NeuTox 䝁䞁䝋䞊䝅䜰䝮

ᅜ㝿

㐃ᦠ

ᚓ䜙䜜䛯ẘᛶ䝕䞊䝍䜔ᩥ⊩᝟ሗ➼䛿

⾜ᨻ䛻ά⏝䛷䛝䜛䜘䛖䛻㐺ᐅሗ࿌

᪂つ in vitro ヨ㦂ἲ䛾ᥦ᱌

JaCVAM

᪂つヨ㦂ἲ䛾ᥦ᱌䜢㋃䜎䛘䛯䝻䞊䝗䝬䝑䝥

ᮏ⏦ㄳ᭩

ᑗ᮶䛾ィ⏬

㐃ᦠ

ᮏ◊✲⌜䛾≉ᚩ

1. in vitro ヨ㦂ἲ☜❧䛻ྥ䛡䛶䚸୍య䛸

䛺䛳䛶ྲྀ䜚⤌䜐 2. ᅜ㝿ⓗ䛺༠ㄪ䜢ᅗ䜛

䛣䜜䜎䛷䛻☜❧䛧䛯ホ౯ἲ䛾୍ぴ

ྠ୍䛾㝧ᛶᑐ↷≀㉁䛻ᑐ䛧䛶䚸⫾⏕ᮇ䛸ᡂ⇍ᮇ䛻䛚䛡䜛స⏝䜢 ẚ㍑䛧䚸ྛⓎ㐩ẁ㝵䛻䛚䛔䛶ẘᛶホ౯䛷䛝䜛䛣䛸䜢♧䛧䛯䚹

⫾⏕䞉⚄⤒Ⓨ㐩ᮇ䛾ホ౯⣔

㻭㻦 䝠䝖㼕㻼㻿⣽⬊䛾⚄⤒⣔ศ໬ホ౯ἲ 㻮㻦 䝷䝑䝖⚄⤒ሐ⣽⬊䛾㐟㏦ホ౯ἲ 㻯㻦 䝷䝑䝖⏕ᚋึᮇ䛾⚄⤒᪂⏕ホ౯ἲ ᡂ⇍ᮇ䛾䝅䝘䝥䝇ᶵ⬟ホ౯⣔

㻰㻦 䝷䝑䝖ᾏ㤿䛾่⃭ᛂ⟅ホ౯ἲ 㻱㻦 䝷䝑䝖ᑠ⬻䛾✺㉳ఙᒎホ౯ἲ 䈜ホ౯䛧䛯Ⓨ㐩᫬ᮇ䛿䚸ᕥᅗ䛾㻭䡚㻱䚹 䈜ホ౯䛧䛯㝧ᛶᑐ↷≀㉁䛿䚸 㻝㻚 䝞䝹䝥䝻㓟䠄㼂㻼㻭䠅 㻞㻚 䝖䝸䝤䝏䝹䝇䝈䠄㼀㻮㼀䠅 㻟㻚 䜽䝻䝹䝢䝸䝩䝇䠄㻯㻼㻲䠅

EST 䛿 2001 ᖺ䛻 ECVAM 䛾ㅎၥᶵ㛵䛛䜙⏕ṪⓎ⏕ẘᛶ 䛾௦᭰ヨ㦂ἲ䛸䛧䛶ᥦ᱌䛥䜜䛯䚹

Embryo Stem cell Test

in vitroヨ㦂ἲ 䛾⤂௓

ES⣽⬊䛾ቑṪ㜼ᐖ

ES⣽⬊䛾ศ໬⬟䠄ᢿື⬟䠅䛾㜼ᐖ 3T3⣽⬊䛾ቑṪ㜼ᐖ

ྛᐇ㦂䛛䜙⟬ฟ䛧䛯IC ₅₀ -3T3䚸IC ₅₀ - ES 䚸 ID 50 -ES 䜢Ỵ䜑䜙䜜䛯ィ⟬ᘧ䛷 ゎᯒ

⿕㦂≀㉁䛾⏕ṪⓎ⏕ẘᛶ䜢ホ౯

䛧䛛䛧䛺䛜䜙䚸OECD 䛾䜺䜲䝗䝷䜲䞁໬䛻䛿⮳ 䜙䛺䛛䛳䛯䚹 䛭䛾⌮⏤䛸䛧䛶䚸

䐟ᵝ䚻䛺ẘᛶ䝯䜹䝙䝈䝮䜢䜒䛴ከ✀㢮䛾⿕㦂≀㉁䛾᳨ド 䐠ุᐃἲ䛾ᨵⰋ

䐡⚄⤒䜔㦵䛺䛹ᚰ➽௨እ䛾⣽⬊䜈䛾ศ໬ㄏᑟ⣔䛾ᑟධ 䐢௦ㅰ䛾ホ౯䛾ᑟධ➼䛾ᚲせᛶ

䛺䛹䚹

Embryo Stem cell Test

ヨ㦂ἲ䛾⤂௓

EST ἲ䛾ᨵⰋ

ண ᛶ䛜䜎䛰㧗䛔䛸䛿䛔䛘䛪䚸䛥䜙䛻ᨵⰋ䛩䜛ᚲせᛶ

⤫ྜⓗ䛺䜰䝥䝻䞊䝏䛜ᚲせ

TOXICOLOGICAL SCIENCES 124(2), 460–471 (2011)

ILSI Health and Environmental Sciences Institute

Activities and Accomplishments

HESI Translation Biomarkers of Neurotoxicity (NeuTox) Committee

2015-2016

ILSI Health and Environmental Sciences

Institute

9

HESI Subcommittee:

Pilot Study Protocol

Main objective: identify circulating biomarkers that predict central & peripheral neurotoxicity resulting from exposure to a known and well-characterized neurotoxic agent by correlating them with behavioral, imaging, morphometric and neuropathological endpoints.

• US FDA NCTR contributing rats, lab space and imaging – timeline Sept – Nov 2015.

• Prototypical compound trimethyltin (TMT).

• Time-course assessments of blood, CSF, CNS, urine and imaging (MRI, MRS) compared to traditional assessment (i.e. functional (behavioral), and histopathology)

• Dosing at 8 mg/kg

• Sample collection, behavioral analyses and MRI/MRS imaging at 2, 6, 10, 14, 21 days

ILSI Health and Environmental Sciences

Institute

10

Additional accomplishments

• Subteam formed to explore possible project to identify seizuregenic compounds using microelectrode array (MEA).

• Session planned at the 2015 Safety Pharmacology Society Annual Meeting in September.

Increases in Autism Spectrum Disorders in USA

(Data from CDC)

'17WHVWLQJ

Ⓨ㐩ẘᛶヨ㦂

Developmental Neurotoxicity Testing for 2,863 Chemicals

Produced Above 1 million pounds/year

21.4%

0.4%

78.2%

No Data On Developmental Toxicity

12 Tested for Neurodevelopmental Toxicity

According to EPA Guidelines Some Data

On Developmental Toxicity

Environmental Health Perspectives, 117:17 (2009)

Current Status of DNT Testing

• Large numbers of chemicals identified for testing (e.g., pesticide) with no risk-based criteria for setting testing priorities

• Different regulatory authorities/different testing requirements with no scientific basis for flexible testing approach

• Current guideline testing is expensive, time consuming and requires large numbers of animals

Research Challenge

• Develop alternative testing approaches that are fast and efficient

– Use in vitro cell culture or in silico models – Use alternative species (non-mammalian)

• Provide data for prioritization of chemicals for further testing (targeted?)

• Such an approach will:

– Reduce costs and animal use

– Facilitate screening of large numbers of chemicals (high- throughput)

Research Approach - In Vitro

• In vitro tests based on key events of CNS development

– proliferation, differentiation, growth, synaptogenesis, myelination, apoptosis

• Endpoints amenable to high throughput testing – cell-based endpoints, biomarkers, molecular signaling

• Show predictive ability based on “training set” of developmental neurotoxicants

Key events of DNT at the cellular level 䝯䜹䝙䝈䝮

䝠䝖iPS⣽⬊䜢⏝䛔䛯

Ⓨ㐩⚄⤒ẘᛶヨ㦂䛾ྍ⬟ᛶ

1. Time, Cost-consuming 2. High Throughput screening 3. Species difference

in vitro test method using human iPS cells

0 4 10

Day KSR

Ectodermal

N2

3D [ 2F W ^'D\

˩P

'D\

˩P

'D\

˩P

Neural Progenitor cells 1HXUDOGLIIHUHQWLDWLRQE\GXDO60$'LQKLELWLRQ

LQKXPDQL36FHOOV

LDN193189 SB431542

DNT compounds

From EPA database

Effect of TBT on neural differentiation

0 1 2 3 4 5 6 7

Pax6

0 0.2 0.4 0.6 0.8 1 1.2

Nanog

L36 (FWRL36 L36 (FWRL36 7%7 (FWRL36

*H QH H [S UH VVL RQ IR OG

7LPHFRXUVHGD\

3$;

YHKLFOH

&3)

Effect of chlorpyrifos on neural differentiation

ኼእነዐኦ዇ቿ❐役丰䚕ቑⓅ㈰

娜⚗ ⒕孑

0IQ )LV'US

ኼእነዐኦ዇ቿ含榊⇜值㖐 ㇱ㏚Ⓟ㈰቎ቫቮ❐役丰䚕 7&$ኒኁኌወ

含栢+㼁ቢ⒉ሺዘ䉒ㄵ▍揜ㇱ㒟

ኼእነዐኦ዇ቿ㳮厌⇝ₚ ኼእነዐኦ዇ቿ㳮厌值㖐ዘℱ拁

+

+

+

+2 2 1$'+

1$' 7&$

ኒኁኌወ

␔含

˂˕

榊⷟↬拣侊

TBT䛻䜘䜛䝭䝖䝁䞁䝗䝸䜰⼥ྜ䝍䞁䝟䜽㉁䛾ศゎ

0 0IQ 0IQ

&2;,9 EDFWLQ

Q07%7KU

& & & 3

Mfn1, 2

Drp1, Fis1

Yamada et al., Metallomics, 2015

FRQWURO

7$

7%7

&&&3

䝠䝖 iPS ⣽⬊䛾䝭䝖䝁䞁䝗䝸䜰ᙧែ䛻ᑐ䛩䜛 䜽䝻䝹䝢䝸䝩䝇䛾ᙳ㡪

FRQWURO &3)

%DU PP

Yamada et al., in preparation

3$; )2;* 1&$0

FRQWURO 0IQ

*H QH H[ SU HV VLRQ I ROG

0IQ 0IQ EDFWLQ

FR QW UR O 0 IQ

Yamada et al., in preparation

䝠䝖iPS⣽⬊䛾⚄⤒ศ໬䛻ᑐ䛩䜛 Mfn1䛾ᙳ㡪

Summary

v Pax6 ⁺ Chlorpyrifos

Tributyltin

Neural progenitor cells

iPS cells Neuron

1. Capability of neural differentiation of human iPS cells can be used for assessment of chemicals with DNT.

2. This approach will reduce animal use and costs, facilitate screening of large numbers of chemicals and might provide data for prioritization of chemicals for further testing.

Oct4 ⁺

ᾏ

ᾏ㤿䝙䝳䞊䝻䞁䜢⏝䛔䛯

⚄⤒䝛䝑䝖䝽䞊䜽䛻䜘䜛ホ౯ἲ䛾㛤Ⓨ

࠯঺28࠰ࡇҽဃі΁Ⴞᅹܖᄂᆮᝲᙀя᣿

ί҄ܖཋឋἼἋἁʙಅὸ

Ẑႆᢋ஖ỆấẬỦወӳႎễ᡿ႆࣱᅕኺ൒ࣱᚾ᬴ඥỉ᧏ႆẑ ᇹ1ׅ ྰ˟ᜭ ί2016࠰9உ2ଐὸ

Ўਃᄂᆮᎍᾉޛ߃ ٻ೔ί׎ᇌҔᕤԼ᫢Լᘓဃᄂᆮ৑ὸ ңщᄂᆮᎍᾉႉރ ୓ଢί፭ᬔٻܖὸ

Ⓨ⏕㐣⛬䜢ᶍೌ䛧䛯⣽⬊

䛻

䛻䜘䜛䝇䜽䝸䞊䝙䞁䜾

䝅䝘䝥䝇ᡂ⇍ᮇ䛻䛚䛡䜛 㐜Ⓨᛶẘᛶ䛾ホ౯

䞉䝥䝻䝖䝁䝹ᩚഛ 䞉AOP䛾ᣦᶆ䜢ᥦ᱌ 䞉᪂つヨ㦂ἲ䛾ᥦ᱌

⤫ྜⓗⓎ㐩⚄⤒ẘᛶヨ㦂䛾☜❧

+(6,1HX7R[

ᅜ㝿㐃ᦠ

ᚓ䜙䜜䛯ẘᛶ䝕䞊䝍䛸

᪤Ꮡ䛾䝠䝖䞉ື≀䝕䞊䝍 䛾᳨ウ

ඹ㏻䛾໬Ꮫ≀㉁䛻䜘䜛᳨ド ᪂つAOP䛾ᥦ᱌

ศᢸ◊✲ㄢ㢟䛻䛴䛔䛶

ᅕኺἋἣỶἁỆợỦᅕኺ෇ѣᚸ̖ඥ䠄◊✲༠ຊ⪅䠖⩌㤿኱Ꮫ䞉ⓑᑿ䠅

ⓑᑿ◊䜘䜚ᥦ౪䛥䜜䜛෾⤖䝷䝑䝖ᾏ㤿䝙䝳䞊䝻䞁䜢⏝䛔䛶䚸䝇䝟䜲䜽䛻䜘䜛䝛䝑䝖䝽䞊䜽ά

ືホ౯䝥䝻䝖䝁䝹䜢ᩚഛ䛧䚸෌⌧ᛶ䛺䛹䜢᳨ド䛩䜛䚹䜎䛯䚸㧗䝇䝹䠉䝥䝑䝖䝇䜽䝸䞊䝙䞁䜾

⣔䛾ᵓ⠏䜢㐍䜑䜛䚹

ᅕኺἋἣỶἁỆợỦᅕኺ෇ѣᚸ̖ඥ䠄◊✲༠ຊ⪅ ⩌㤿኱Ꮫ ⓑᑿ䠅

䛩䛷䛻㛤Ⓨ䛧䛯⏕ᚋ䛾Ⓨ㐩⚄⤒ẘᛶ䛾ホ౯ᡭἲ䛻ᇶ䛵䛝䚸ᅜ㝿䝁䞁䝋 䞊䝅䜰䝮䛸䛾༠ㄪ䚸᪤Ꮡ䛾ẘᛶ䝕䞊䝍䛸䛾ẚ㍑䛻䜘䜚䚸ヨ㦂ἲ䛾᭷ຠ

ᛶ䞉ண ᛶ䜢᳨ド䛧䚸⤫ྜⓗ䛺ヨ㦂ἲ䜢☜❧䛩䜛䚹

ᐇ㦂ᮦᩱ䛻䛴䛔䛶

•

⫾⏕18᪥䛾䝷䝑䝖⫾௘䛛䜙ᾏ㤿⚄⤒⣽⬊䜢༢㞳䛧䚸෾⤖䛧䛯䜒䛾䚹

• 1 tube䛒䛯䜚⣙80୓ಶ䛾⣽⬊䛜ධ䛳䛶䛔䜛䚹

•

⩌㤿኱Ꮫ䞉ⓑᑿ◊䜘䜚ᥦ౪䛔䛯䛰䛔䛶䛔䜛䚹

100 μm

MAP2 drebrin MAP2/drebrin

21᪥㛫ᇵ㣴䛧䛯㝿䛾MAP2䛸drebrin䛾Ⓨ⌧

෗┿ᥦ౪䠖ⓑᑿඛ⏕

SKY Neuron

SKY Neuron䛾ゎ෾

1. ๓᪥䜎䛷䛻mestro䝥䝺䞊䝖䜢PEI䛒䜛䛔䛿PLL䛷䝁䞊䝔䜱䞁䜾

2. ⣽⬊䛾ゎ෾1᫬㛫๓䛻䚸5 μl䛾䝷䝭䝙䞁䠄plating medium䛻⁐ゎ䠅䜢ྛ䜴䜵䝹䛾㟁ᴟ㒊

ศ䛻஌䛫䛶1 hr䜲䞁䜻䝳䝧䞊䝍䞊ෆ䛻㟼⨨

3. 3ศ㛫 ᾎ䛷ゎ෾

4.

䝞䜲䜰䝹୰䛾⣽⬊ᠱ⃮ᾮ䠄1 ml䠅䜢50 ml䝁䝙䜹䝹䝏䝳䞊䝤䛻⛣䛧䚸䝞䜲䜰䝹䜢1 ml

plating medium䛷䝸䞁䝇䛧䚸2䡚5sec/1⁲䛪䛴ῧຍ

5. 2 ml plating medium䜢2~3sec/1⁲䛪䛴ῧຍ

6. 2 ml plating medium䜢ᑡ䛧䛪䛴ῧຍ䛧䚸䜖䛳䛟䜚䛸㌿ಽΰ࿴䞉䞉䞉final vol. 6ml 7.

⣽⬊ᠱ⃮ᾮ䛸䝖䝸䝟䞁䝤䝹䞊䜢䠍䠖䠍䛷ΰ䛬䚸㐺㔞䜢⾑⌫ィ⟬┙䛻ῧຍ䛧⣽⬊ᩘィ 

8. ⣽⬊ᠱ⃮ᾮ䜢15 ml䝁䝙䜹䝹䝏䝳䞊䝤䛻⛣䛧⣽⬊䜢㐲ᚰ䠖ք180 x g, 5min, ᐊ  9. Supᤞ䛶䚸䝍䝑䝢䞁䜾3~4ᅇ䛧䛶䜋䛠䛩

10. 䜹䜴䞁䝖䛧䛯⏕⣽⬊ᩘ䜢ඖ䛻ᠱ⃮䛩䜛ᇵᆅ㔞䜢ィ⟬䛧䚸⣽⬊䜢ᠱ⃮

11. 5 μl䛾⣽⬊ᠱ⃮ᾮ䜢䝁䞊䝖䛧䛯䝷䝭䝙䞁ୖ䛻஌䛫䜛 12. 䝥䝺䞊䝖䛾࿘ᅖ䛻஝⇱䜢㜵Ṇ䛩䜛䛯䜑䛾⁛⳦Ỉ䜢ῧຍ 13. 37Υ, CO

2

incubate, 1 hr.

14. ྛ䜴䜵䝹䛻500 μl䛾Plating medium䜖䛳䛟䜚䛸ῧຍ 16. Incubate, 37Υ, 4days

17. Day4: Ara C䜢culture medium䛷⁐ゎ䛧ᇵᆅ஺᥮䜢஺᥮

19. Incubate, 37Υ

20. Day7䛷ᇵᆅ஺᥮䚸䛭䛾ᚋ3-4᪥䛒䜛䛔䛿7᪥䛚䛝䛻ᇵᆅ஺᥮

21.  ᐃ

ⓑᑿ◊䜘䜚ᥦ౪䛥䜜䛯䝥䝻䝖䝁䝹䜢ᨵኚ

㐲ᚰ᮲௳䛾᳨ウ

ᢒ࣎ẝụί715 x g, 2Ў) ᢒ࣎ễẲ

෗┿ᥦ౪䠖ᑠ㔠⃝ඛ⏕䠄ⓑᑿ◊䠅

ゎ෾᫬䛾⣽⬊⏕Ꮡ⋡

/27 便

便卭㟿

WXEH OLYLQJFHOOV GHDGFHOOV 9LDELOLW\ WRWDOFHOOV

5(+ ™

FHOOVWXEH

5(+ ™

FHOOVWXEH [

FHOOV [

FHOOV [

FHOOV 5(+ ™

FHOOVWXEH [

FHOOV [

FHOOV [

FHOOV 5(+ ™

FHOOVWXEH [

FHOOV [

FHOOV [

FHOOV

5(+ ™

FHOOVWXEH

5(+ ™

FHOOVWXEH [

FHOOV [

FHOOV [

FHOOV 5(+ ™

FHOOVWXEH

5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH

5(+ ™

FHOOVWXEH [

FHOOV [

FHOOV [

FHOOV 5(+ ™

FHOOVWXEH

5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH 5(+ ™

FHOOVWXEH

[

FHOOV [

FHOOV [

FHOOV

[

FHOOV [

FHOOV [

FHOOV [

FHOOV [

FHOOV

[

FHOOV

䞉⏕Ꮡ⋡䛿ẖᅇ50%⛬ᗘ䛷Ᏻᐃ䛧䛶䛔䜛䚹 䞉ⓑᑿ◊䛷䛾⣽⬊ᩘ䜹䜴䞁䝖䛸䛾䜼䝱䝑䝥䛜኱䛝䛔䚹

Maestro system 1

Hippocampus neurons are cultured in 48 well MEA plates

Spontaneous and stimulated activity

McConnell et al., NeuroToxicology, 2012䜢ᨵኚ

Activity map

Continuous Plots

Spike Plots

Maestro system 2

䝇

䝇䝟䜲䜽䛾ホ౯⣔䛻䛴䛔䛶

Mack et al., NeuroToxicology, 2014

ᐇ㦂䛾䝍䜲䝮䝇䜿䝆䝳䞊䝹

DIV -3῍-1

Day 0 4 7 10 14 17 21 24 28

˺ಅ᪮Ⴘ

ἅὊἘỵὅἂίPLL or PEIὸ LamininἅὊἘỵὅἂᚐϵὉછᆔ AraCชьί0.5 or 1 μMὸẆίMaestroยܭ?ὸ MaestroยܭẆίؔעʩ੭ὸ

MaestroยܭẆίؔעʩ੭ὸ MaestroยܭẆίؔעʩ੭ὸ MaestroยܭẆίؔעʩ੭ὸ MaestroยܭẆίؔעʩ੭ὸ Maestro ยܭẆίؔעʩ੭ὸ MaestroยܭẆίؔעʩ੭ὸ

୴ଐ உ ້

᣿ ם உ ້ ங ᣿ உ ້ ங ᣿ உ ້ ங ᣿ உ ້

ὉPLLẝỦẟỊPEIἅὊἘỵὅἂẲẺࢸỊʑ༞ẰẶϬᔺࡉỆ ề̬܍ӧᏡ

ὉmaestroἩἾὊἚồછᆔỊဃኬᏘૠử੔ဇẲềẟỦ ὉἂἼỴኬᏘỉف഻ử৮СẴỦẺỜỆAraCửชьẲềẟỦ ὉᅕኺኬᏘỊภࡇ٭҄ỆݣẲࢊẟẺỜẆؔ᫱ܴẦỤ᬴ܱܴ

ồỉᆆѣỆỊỽἽἮἕἁἋửဇẟềภࡇ٭҄ửಊщᢤẬ

ềẟỦ

ỽἽἮἕἁἋ

䝥䝻䝖䝁䝹ᩚഛ

౨᚛ʙ᪮

Shafer’s lab +Axion protocol ίCortical neuronὸ

வˑṞ ίHippocampusὸ

வˑṟ ίHippocampusὸ ἅὊἘỵὅἂ 0.05% PEIэ

50 μg/ml laminin

0.05% PEIэ 50 μg/ml laminin

50 μg/ml PLLэ 50 μg/ml laminin ኬᏘછᆔ݅ࡇ 150,000 cells/25 μl 25,000 cells/5 μl 50,000 cells/5 μl

AraCຜࡇ 5 μM 0.5 μM 1 μM

APVชь ໯ ໯ ஊ

ؔעʩ੭᣽ Ҟ᣽ʩ੭ Ҟ᣽ʩ੭ μ᣽ʩ੭

ؔעʩ੭ἋἃἊἷὊἽ 7ଐ൑ 3-4ଐ൑ 7ଐ൑

৲ɨ૾ඥ AcuteίDay 12 to 22ὸ Acute Chronic

ᩓൢХນਤዓ଺᧓ ὼ 100 μS 200 μS

Хນᩓಊỉˮፗ ὼ ỸỹἽắểỆ٭҄ μỸỹἽӷẳ

౨᚛ຜࡇૠ 1ຜࡇ

ยܭਦ೅

Mean firing rate Active electrode ί< 5 spikes/minὸ MFR on AE

Mean firing rate

Active electrodeί< 5 spikes/minὸ MFR on AE

Spike counts at stimulation

᮲௳᳨ウ 䠉䝁䞊䝔䜱䞁䜾䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ ṞPLL

ṟPEI ኬᏘછᆔ݅ࡇ

2.5x10⁴cells

AraCຜࡇ 1μM

APVชь

໯

ؔעʩ੭᣽

1/3᣽ʩ੭

ؔעʩ੭

7ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊ ỸỹἽắểỆ

٭҄

1ỸỹἽẝẺụ1Ў᧓ỉዮἋἣỶἁૠ ỴἁἘỵἨễᩓಊૠ

1ᩓಊẝẺụ1Ў᧓ỉἋἣỶἁૠ ᩓൢХນ଺ỆႆဃẲẺἋἣỶἁૠ

PEI PLL

>

᮲

᮲௳᳨ウ 䠉䝁䞊䝔䜱䞁䜾䠉

Day 21

PLL PEI

᮲௳᳨ウ 䠉⣽⬊᧛✀ᐦᗘ䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PEI

ኬᏘછᆔ݅ࡇ Ṟ2.5x10

⁴cells

ṟ5.0x10

⁴cells AraCຜࡇ 0.5 μM

APVชь

໯

ؔעʩ੭᣽

1/3᣽ʩ੭

ؔעʩ੭

7ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊˮፗ ỸỹἽắểỆ

٭҄

1ỸỹἽẝẺụ1Ў᧓ỉዮἋἣỶἁૠ ỴἁἘỵἨễᩓಊૠ

1ᩓಊẝẺụ1Ў᧓ỉἋἣỶἁૠ ᩓൢХນ଺ỆႆဃẲẺἋἣỶἁૠ

2.5 x 10 ⁴ cells 5.0 x 10 ⁴ cells

>

᮲௳᳨ウ 䠉AraC⃰ᗘ䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PEI

ኬᏘછᆔ݅ࡇ

2.5x10⁴cells AraCຜࡇ

Ṟ0.5 μM

ṟ1

μM

APVชь

໯

ؔעʩ੭᣽ μ᣽ʩ੭

ؔעʩ੭

3-4ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊˮፗ ỸỹἽắểỆ

٭҄

1ỸỹἽẝẺụ1Ў᧓ỉዮἋἣỶἁૠ ỴἁἘỵἨễᩓಊૠ

1ᩓಊẝẺụ1Ў᧓ỉἋἣỶἁૠ ᩓൢХນ଺ỆႆဃẲẺἋἣỶἁૠ

0.5 μM AraC 1 μM AraC

>

᮲௳᳨ウ 䠉APVῧຍ䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PEI

ኬᏘછᆔ݅ࡇ

2.5x10⁴cells AraCຜࡇ 0.5 μM

APVชь

Ṟஊ

ṟ໯

ؔעʩ੭᣽ μ᣽ʩ੭

ؔעʩ੭

3-4ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊˮፗ ỸỹἽắểỆ

٭҄

1ỸỹἽẝẺụ1Ў᧓ỉዮἋἣỶἁૠ ỴἁἘỵἨễᩓಊૠ

1ᩓಊẝẺụ1Ў᧓ỉἋἣỶἁૠ ᩓൢХນ଺ỆႆဃẲẺἋἣỶἁૠ

APV- APV+

>

᮲௳᳨ウ 䠉ᇵᆅ஺᥮㔞䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PEI

ኬᏘછᆔ݅ࡇ

2.5x10⁴cells AraCຜࡇ 0.5 μM

APVชь

໯

ؔעʩ੭᣽ ṞҞ᣽ʩ੭ ṟμ᣽ʩ੭

ؔעʩ੭ 3-4ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊˮፗ ỸỹἽắểỆ

٭҄

1ỸỹἽẝẺụ1Ў᧓ỉዮἋἣỶἁૠ ỴἁἘỵἨễᩓಊૠ

1ᩓಊẝẺụ1Ў᧓ỉἋἣỶἁૠ ᩓൢХນ଺ỆႆဃẲẺἋἣỶἁૠ

༙㔞஺᥮

඲㔞஺᥮

>

᮲௳᳨ウ 䠉㟁Ẽ่⃭ᣢ⥆᫬㛫䠉

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PLL

ኬᏘછᆔ݅ࡇ

2.5 x 10⁴cells

AraCຜࡇ 1μM

APVชь

໯

ؔעʩ੭᣽

1/3᣽ʩ੭

ؔעʩ੭

7ଐ൑

ᩓൢХນ଺᧓ Ṟ100μS ṟ200μS Хນᩓಊˮፗ μỸỹἽӷẳ

REH1004

REH1006

100 μS 200 μS

=

᮲

᮲௳᳨ウ 䠉่⃭㟁ᴟ఩⨨䠉

㻰㼍㼥㻌㻣 㻰㼍㼥㻝㻜 㻰㼍㼥㻞㻝 㻰㼍㼥㻞㻠 㻰㼍㼥㻞㻤

㻰㼍㼥㻣 㻥㻣㻚㻞

㻰㼍㼥㻝㻜 㻝㻞㻟㻚㻢 㻝㻝㻢㻚㻢

㻰㼍㼥㻝㻠 㻝㻞㻣㻚㻤 㻝㻜㻥㻚㻞

㻰㼍㼥㻝㻣 㻝㻠㻜㻚㻢 㻝㻝㻢㻚㻞

㻰㼍㼥㻞㻝 㻝㻣㻠㻚㻢 㻞㻝㻝㻚㻞 㻝㻤㻤

㻰㼍㼥㻞㻠 㻝㻢㻟㻚㻢 㻝㻟㻜㻚㻤 㻝㻤㻜㻚㻤 㻝㻥㻣㻚㻤

㻰㼍㼥㻞㻤 㻝㻠㻠㻚㻤 㻝㻥㻝㻚㻞 㻞㻠㻡 㻝㻠㻡㻚㻤 㻝㻣㻟㻚㻞

㻞㻚㻡㻌㼤㻌㻝㻜^㻠㻘 㻼㻱㻵㻘㻌㻭㻼㼂㻗㻘 㻝㻌㻭㼞㼍㻯

ٴٴٴ ٴ ٴٴ ٴ ٴ ٴ ٴ ٴٴ ٴٴ ٴ ٴ

ٴٴٴ ٴ ٴٴ ٴ ٴ ٴٴٴ ٴ ٴٴ ٴ ٴ

ٴٴٴ ٴ ٴٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴٴ ٴٴ

ٴٴٴ ٴ ٴٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴٴ ٴٴ

ٴٴٴ ٴ ٴٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴٴٴٴ

㻝㻜㻜㼡㼟 㻭㻢

ٴٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴ ٴٴ ٴ ٴ

䖃䠖่⃭㟁ᴟ

ὉἋἣỶἁầЈྵẲềẟễẟᩓಊỊẆኬᏘầʈẾềẟễẟểЙૺẰủỦẺỜХນửẲềờ ӒࣖẲễẟểᎋảỤủỦẇэỸỹἽắểỆ٭ảỦỔẨẇ

ὉἋἣỶἁЈྵᩓಊỉХນˮፗử٭ảềờӒࣖỉᆉࡇỊٻẨẪ٭҄ẲễẦẾẺẇ

э1ࡇܭỜềẲộảịẆยܭଐ൑Ệ٭ảỦ࣏ᙲỊễẟỉỂỊễẟẦẇ

౨᚛ʙ᪮ வˑ

ἅὊἘỵὅἂ

PLL

ኬᏘછᆔ݅ࡇ

2.5 x 10⁴cells

AraCຜࡇ 1μM

APVชь

໯

ؔעʩ੭᣽ μ᣽ʩ੭

ؔעʩ੭

3-4ଐ൑

ᩓൢХນ଺᧓

100μS

Хນᩓಊˮፗ ṞỸỹἽắểỆ

٭҄

ṟμỸỹἽӷẳ

䜴䜵䝹䛤䛸䛻ኚ໬

඲䜴䜵䝹ྠ䛨

>

Ỵᐃ᮲௳䛾䜎䛸䜑

౨᚛ʙ᪮

Shafer’s lab +Axion protocol ίCortical neuronὸ

வˑṞ

ίHippocampusὸ வˑṟ ίHippocampusὸ ἅὊἘỵὅἂ 0.05% PEIэ

50 μg/ml laminin

0.05% PEIэ 50 μg/ml laminin

50 μg/ml PLLэ 50 μg/ml laminin ኬᏘછᆔ݅ࡇ 150,000 cells/25 μl 25,000 cells/5 μl 50,000 cells/5 μl

AraCຜࡇ 5 μM 0.5 μM 1 μM

APVชь ໯ ໯ ஊ

ؔעʩ੭᣽ Ҟ᣽ʩ੭ Ҟ᣽ʩ੭ μ᣽ʩ੭

ؔעʩ੭ἋἃἊἷὊἽ 7ଐấẨί1/2ὸ 3-4ଐấẨί1/2ὸ 7ଐấẨί1/2ὸ

৲ɨ૾ඥ AcuteίDay14Ệềยܭὸ Acute Chronic

ᩓൢХນਤዓ଺᧓ ὼ 100 μS 200 μS

Хນᩓಊỉˮፗ ὼ ỸỹἽắểỆ٭҄ μỸỹἽӷẳ

౨᚛ຜࡇૠ 1ຜࡇίDay14Ệềยܭὸ

ยܭਦ೅

Mean firing rate Active electrode ί< 5 spikes/minὸ MFR on AE

Mean firing rate

Active electrodeί< 5 spikes/minὸ MFR on AE

Spike counts at stimulation

௒ᚋ䛾ணᐃ

ὉൿܭẲẺவˑỆềẆᙐૠỉ҄ܖཋឋửဇẟẺᚸ̖ửᡶỜỦẇ Ὁᚸ̖ẴỦ҄ܖཋឋỉᢠܭὉ৲ɨ૾ඥὉຜࡇൿܭửᘍạẇ

ί᛻ဋྰỆềܱጚỉẝỦ҄ܖཋឋẦỤܱ଀ẴỦỉầ໯ᩊ?ὸ

ίἋἻỶἛ22-23, HESI NeuTox҄ӳཋӏỎἋἻỶἛ24, ShaferỤầ੔ဇẲềẟ Ủ҄ӳཋἼἋἚờஊụὸ

ὉmaestroἙὊἑẦỤࢽỤủỦૼẺễਦ೅ỆợỦᚐௌửᘍẟẆợụ ችࡇὉʖยࣱỉ᭗ẟᚐௌඥửᢠܭẴỦẇ

ίἋἻỶἛ25Ӌༀὸ

ὉEPAỉDr. Timothy J. Shaferể᬴ܱኒấợỎ᬴ܱἙὊἑỆếẟ ềỉऴإσஊửᘍẟẆଏ܍ỉ൒ࣱἙὊἑểỉൔ᠋ửᘍạẇ ί9/30ỆEPAỆề৙ẼӳỪẶỉʖܭὸ

Ὁ˂ЎਃᎍỉኽௐሁểወӳႎỆྸᚐỂẨỦợạᡲઃẲềᡶỜỦẇ

HESI NueTox䛻䛚䛡䜛ホ౯໬ྜ≀

Neurotoxicants/

Dev. Neurotoxicants

Flame Retardants

Polycyclic Aromatic

Hydrocarbons Unknowns

1-methyl-4-phenylpyridinium iodide 2- ethylhexyl diphenyl phosphate (EHDP) 4-H-Cyclopenta(d,e,f)phenanthrene 1-ethyl-3-methylimidazolium diethylphosphate

2-Methoxyethanol 2,2',4,4',5,5'-Hexabromodiphenyl ether Acenaphthene Berberine chloride

3,3'-Iminodipropionitrile 2,2',4,4',5-Pentabromodiphenyl ether Acenaphthylene Carbamic acid, butyl-, 3-iodo-2-propynyl ester

5-Fluorouracil 2,2'4,4'-Tetrabromodiphenyl ether Anthracene Manganese, tricarbonyl[(1,2,3,4,5-.eta.)-

6-Hydroxydopamine hydrochloride 2,3,7,8-Tetrachlorodibenzo-p-dioxin Benz(a)anthracene 1-methyl-2,4-cyclopentadien-1-yl]-

6-Propyl-2-thiouracil 3,3’,5,5’-Tetrabromobisphenol A Benzo(a)pyrene

Acetic acid, manganese(2+) salt Isodecyl diphenyl phosphate Benzo(b)fluoranthene

Acrylamide Phenol, isopropylated, phosphate (3:1) Benzo(e)pyrene

Aldicarb tert-Butylphenyl diphenyl phosphate Benzo(k)fluoranthene

Bis(tributyltin)oxide Tricresyl phosphate Benzo[g,h,i]perylene

Bisphenol A Triphenyl phosphate Chrysene

Captan Tris(2-chloroethyl) phosphate Dibenz(a,h)anthracene

Carbaryl Bis(2-ethylhexyl) 3,4,5,6- tetrabromophthalate (TBPH) Dibenz[a,c]anthracene Chlorpyrifos (Dursban) 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) Fluorene

Colchicine tris(2-Chloroisopropyl)phosphate, TCPP Naphthalene

Deltamethrin Firemaster-550 Phenanthrene

Di(2-ethylhexyl) phthalate Pyrene

Diazepam DDT Dieldrin Diethylstilbestrol Heptachlor Hexachlorophene Hydroxyurea Lindane Methyl mercuric (II) chloride n-Hexane Parathion Permethrin Phenobarbital sodium salt Rotenone Tebuconazole Tetraethylthiuram disulfide Thalidomide Toluene Valinomycin Valproic acid sodium salt

Negative Controls

Acetaminophen Acetylsalicylic acid D-Glucitol L-Ascorbic acid Saccharin Sodium Salt hydrate

Other NTP Compounds

Bisphenol A Bisphenol AF Bisphenol S

Neurotoxicants/Dev. Neurotoxicants in HESI NeuTox

1-methyl-4-phenylpyridinium iodide 2-Methoxyethanol

3,3'-Iminodipropionitrile 5-Fluorouracil

6-Hydroxydopamine hydrochloride 6-Propyl-2-thiouracil

Acetic acid, manganese(2+) salt Acrylamide

Aldicarb Bis(tributyltin)oxide Bisphenol A Captan Carbaryl

Chlorpyrifos (Dursban) Colchicine Deltamethrin Di(2-ethylhexyl) phthalate Diazepam

DDT

Dieldrin Diethylstilbestrol Heptachlor Hexachlorophene Hydroxyurea Lindane

Methyl mercuric (II) chloride n-Hexane

Parathion Permethrin

Phenobarbital sodium salt Rotenone

Tebuconazole Tetraethylthiuram disulfide Thalidomide

Toluene Valinomycin Valproic acid sodium salt

Shafer’s lab䛻䛶᥇⏝䛧䛶䛔䜛໬ྜ≀䝸䝇䝖

᛻ဋྰỂ̅ဇܱጚẝụ ᛻ဋྰỂ̅ဇܱጚỊễẟầẆಊỜề˩Ẻཋឋ

McConnell et al., NeuroToxicology, 2012

ᢞ୚⃰ᗘ䠖50

μM

᪂

᪂つホ౯ᣦᶆ䛾ೃ⿵

Cotterill et al., J Biomol Screen, 2016

࠯

࠯঺࠰ࡇҽဃі΁ᅹܖᄂᆮᝲᙀя᣿҄ܖཋ ឋȪǹǯᄂᆮʙಅ

žႆᢋ஖ƴƓƚǔወӳႎƳ᡿ႆࣱᅕኺ൒ࣱᚾ᬴

ඥƷ᧏ႆſ

࠯঺࠰ࡇ ᇹׅྰ˟ᜭ

ᄂᆮЎਃᎍᾉɥ᣼ ା

ίငಅҔٻ ငಅဃ७ᅹܖᄂᆮ৑ Ꮀಅࣱɶ൒ܖὸ ᄂᆮңщᎍᾉᇳဋဌኔ܇

ᵆငಅҔٻ ငಅ̬ͤܖᢿ ˺ಅ࿢ؾᚘยСࣂܖὸ

࠯঺ᵐᵖ࠰ᵗஉᵐଐί᣿ὸο᣻ෂ͕ಏᢿ ᇹᵕ˟ᜭܴ

ෙᬔƱƸ ^{¾᪽ͨᓶỉϋᢿỆẝỦ ¾ᚡচỉӮˋذ}

¾ٻᏯႝឋểᅕኺᡲዂầẝỦ ଐࠝႎễЈஹʙởᙾảẺऴإሁ ỊẆෙᬔỉɶỂẟẾẺỮἧỳỶἽẰ ủૢྸૢ᪶ẰủỦẇẸỉࢸٻᏯႝ

ឋỆᔛᆢẰủềẟẪẇ

¾ऴѣỆ᧙ẴỦᏯϋׅែỉɟᢿ

¾ἻἕἚỉෙᬔờᚡচỂ᣻ᙲ

SCIENCE COUNCIL OF JAPAN

ἻἕἚỉෙᬔ

•

ෙᬔỉޖನᡯỊᵐếỉᾤ܌ầӼẨӳẾẺޖನᡯίኬ Ꮨ݅ࡇầٻẨẟỉỂ᱅ẟዴỉợạỆᙸảỦὸẇ

•

ෙᬔỉޖನᡯỊẆἤἚểἻἕἚỂỊ˩ềẟỦẇ

ἻἕἚỉෙᬔỉૺ᩿׋

ἤἚỉෙᬔỉૺ᩿׋

ᵡᶐᵿᶇᶅᴾᵵᵿᶒᶑᶍᶌίޛϋଯᨺ ᚪὸẐὁἚἏὅ ᅕኺᚐаܖỴἚἻἋẑᵆᵏᵗᵗᵓᵇἳἙỵỽἽὉ ἇỶỺὅἋὉỶὅἑὊἜἉἹἜἽ ᵮᵿᶖᶇᶌᶍᶑᵊᴾᵲᶆᶃᴾᵰᵿᶒᴾᵬᶃᶐᶔᶍᶓᶑᴾᵱᶗᶑᶒᶃᶋᴾ ᵱᶃᶁᶍᶌᶂᴾᵣᶂᶇᶒᶇᶍᶌᴾᵆᵏᵗᵗᵓᵇᴾᵟᶁᵿᶂᶃᶋᶇᶁᴾ ᵮᶐᶃᶑᶑ

ȩȃȈෙᬔǹȩǤǹ೅ஜ

Skutella & Nitsch, 2001

঺ྎậẾഫ᫏ỉෙᬔ˳ϋ἟ἕἚὁὊἁ

¾ ෙᬔỊᆃࡀẻẾẺኬᏘನሰửਤếίἤἚể᫏˩ὸ

¾ ෙᬔϋỉᅕኺ἟ἕἚὁὊἁỊẴỂỆᄂᆮẰủềẟỦᵆɦ׋ὸ

¾ ấờễᐻڠࣱἉἜἩἋˡᢋཋឋỊἂἽἑἱὅᣠᵆἤἚể᫏˩ᵇ

¾ ấờễ৮СࣱἉἜἩἋˡᢋཋឋỊᵥᵟᵠᵟίἤἚể᫏˩ᵇ

¾ ᩓൢဃྸܖႎᄂᆮẆᕤྸܖႎᄂᆮỆợẪ̅ဇẰủỦ

ᚸ̖଺஖

ဃࢸᵏᵑḗᵏᵖଐᱫỉἻἕἚෙᬔ

• ἉἜἩἋ࢟঺஖ỆẝẺỦဃࢸᵏᵑḗᵏᵖ ଐᱫỉᏯỊἤἚỂỊဃࢸᵏḗᵐ࠰ỆႻ

࢘ẴỦẇ

• ᢿˮỆợỦࠀầẝỦể࣬ỪủỦầẆ ᏯểẲềỊᐻڠኒὉ৮СኒầểờỆ ٻẨẪ٭҄ẴỦ଺஖ỂẝỦẇ

Ẑᵬᶃᶓᶐᵿᶊᴾᵡᶇᶐᶁᶓᶇᶒᴾᵢᶃᶔᶃᶊᶍᶎᶋᶃᶌᶒᴾᵿᶌᶂᴾᵤᶓᶌᶁᶒᶇᶍᶌᴾᶇᶌᴾᶒᶆᶃᴾᵦᶃᵿᶊᶒᶆᶗᴾᵿᶌᶂᴾ ᵢᶇᶑᶃᵿᶑᶃᶂᴾᵠᶐᵿᶇᶌẑợụ

ᄂᆮƷႸႎ

„ Ꮢဃ஖Ệ҄ܖཋឋử৲ɨẲẺˀἻἕἚỆếẟềẆଐᱫᵏᵑଐẦỤᵏᵖ ଐỆấẬỦෙᬔỉᅕኺׅែೞᏡử౨᚛ẲẆ᡿ࡨࣱႆᢋᅕኺ൒ࣱ

ỉᚸ̖ỆếễầỦਦ೅ử੕ኧẲẆᶇᶌᴾᶔᶇᶒᶐᶍᚾ᬴ඥỂỉኽௐểӳỪ ẶềૼẲẟᚾ᬴ඥỉ᧏ႆỆኽỎ˄ẬỦẇ

vaginal plugs/smears

GD 0 15

chemical administration Pregnant dams

21

Pups (in lactation period)

0 20

Hippocampal slice preparation

Stimulus-response relationship

13 14 15 16 17 18 PND

14 15 16 17 Effect of BMI on PS

PND Evaluation

PND

ෙᬔᅕኺȍȃȈȯȸǯᲴႆᢋᅕኺ൒ࣱᚸ̖

ƷƨNJƷȗȭȈdzȸȫ

ኬ

ኬᏘٳᚡ᥵ඥƷཎࣉ

„

ኬᏘٳᚡ᥵ඥểỊẆ࠼፯ỆỊᅕኺኬᏘᡈͯỆဃẳỦࣇࢊễᩓൢႎẝỦẟỊᩓ

ൢ҄ܖႎ٭҄ửẒᩓئᩓˮẓểẲềᛦỔỦᚡ᥵ඥẇ

„

ႸႎỉᢿˮỆยܭᩓಊίỾἻἋࣇݱᩓಊὸửХλẲẆҗЎỆᩉủềᩓൢႎ٭ѣ ỉݲễẟᢿˮỆؕแᩓಊίɧ᧙ᩓಊὸửᚨẬỦểẆᅕኺኬᏘỉᨼׇ෇ѣỉኽௐ ဃẳỦޅ৑ỉἧỵὊἽἛᩓˮửᚡ᥵ẴỦẮểầỂẨỦẇ

„

ཎࣉ

Ṟ ٶẪỉᅕኺኬᏘỉᩓൢႎễ٭҄ửӷ଺ỆểỤảỦίӷ஖ࣱὸẮểầỂẨỦỉ ỂẆޅ৑ỉᩓൢႎễࣱឋầỪẦỦẇ

ṟ ᅕኺኬᏘᐏử්ủỦỶỼὅᩓ්ỉዮᚘửᩓئᩓˮểẲềểỤảỦẇෙᬔỆ ếẟềỊဃྸܖႎễᛯྸỂᚐ᣷ỊӧᏡỂẆᕤྸܖႎỆờᄩᛐẴỦẮểầ ỂẨỦẇ

Ṡ ᵧᶌᶒᵿᶁᶒễἋἻỶἋ೅ஜử˺঺ỂẨủịẆ২ᘐႎễᩊତࡇỊ˯ẟẇ

ᵏ̾ỉἉἜἩἋẦỤႆဃẴỦ ᩓˮởᵏ̾ỉᅕኺኬᏘẦỤ

ႆဃẴỦ෇ѣᩓˮ

ᅕኺኬᏘᐏử

්ủỦᩓ් ίኬᏘٳὸᩓئᩓˮ ҥɟᅕኺኬᏘἾἫἽ

ᅕኺኬᏘỉᨼׇ

ኬᏘٳᚡ᥵ᩓˮƷဃྸܖႎᚐ᣷

ᨼӳἋἣỶἁᩓˮ ᵮᶍᶎᶓᶊᵿᶒᶇᶍᶌᴾᶑᶎᶇᶉᶃ ᵆᵮᵱᵇửᚡ᥵

ᨼӳᐻڠࣱἉἜἩἋࢸ ᩓˮᵆᶄᶇᶃᶊᶂᴾᶃᶖᶁᶇᶒᵿᶒᶍᶐᶗᴾ ᶎᶍᶑᶒᶑᶗᶌᵿᶎᶒᶇᶁᴾ ᶎᶍᶒᶃᶌᶒᶇᵿᶊᵆᶄᵣᵮᵱᵮᵇửᚡ᥵

Хນᩓಊ

ܱ˳᫋ࣇᦟɦỆᚇݑẰủỦෙᬔἋἻỶἋể ᵐஜỉᚡ᥵ᩓಊểХນᩓಊίዯὸ

500Pm

ᩓˮỉᚐ᣷

ᵏᵌ ᅕኺኬᏘẦỤỉЈщཎࣱ

•

ᨼӳἋἣỶἁᩓˮửᚡ᥵ẲềẆἋἣỶἁᩓˮ ỉਰࠢί᩷ዴὸửᚐௌẴỦ

•

෇ѣᩓˮửႆဃẲềẟỦኬᏘỉૠửӒପ ᵐᵌ ᅕኺኬᏘồỉλщཎࣱ

•

ᨼӳᵣᵮᵱᵮửᚡ᥵ẲềẆẸỉͼẨί᩷ዴὸửᚐ

ௌẴỦ

•

Ẓᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃẓểẟạ

•

ᐻڠࣱἉἜἩἋỉࢍẰửӒପ

ᨼӳἋἣỶἁᩓˮ

ᵮᶍᶎᶓᶊᵿᶒᶇᶍᶌᴾᶑᶎᶇᶉᶃ ᵆᵮᵱᵇ

ᨼӳᐻڠࣱἉἜἩἋ ࢸᩓˮᵆᨼӳᵣᵮᵱᵮᵇ ᵆᵤᶇᶃᶊᶂᴾᶃᶖᶁᶇᶒᵿᶒᶍᶐᶗᴾ ᶎᶍᶑᶒᶑᶗᶌᵿᶎᶒᶇᶁᴾ ᶎᶍᶒᶃᶌᶒᶇᵿᶊᵊᴾᶄᵣᵮᵱᵮᵇ

ਰࠢ

ͼẨ

ХນࣖሉࣱƴǑǔᚸ̖

s tim u la tio n (PA )

P S a m p li tu d e ( mV )

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 0

2 4 6 8 1 0 1 2

s tim u la tio n (PA )

fE PS P s lo p e ( m V /m s )

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 0

2 4 6 8 1 0 1 2

¾ ᵮᵱਰࠢấợỎᶄᵣᵮᵣᵮ ᶑᶊᶍᶎᶃỉٻẨẰỉᩓൢХນ̔܍ࣱửᚸ̖ẴỦẇ

¾ ̊ảịӷẳᩓൢХນỂờࣖሉࣱầفٻẲềẟủịẆᅕኺׅែỆấẬỦᐻ ڠࣱλщίᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃὸẝỦẟỊᐻڠࣱЈщίᵮᵱਰࠢὸầفࢍẲềẟỦẮ ểửᅆẴẇ

¾ ဃࢸᵏᵑଐᱫẦỤᵏᵖଐᱫộỂỉଐᱫắểỉХນࣖሉࣱẆễỤỎỆẸỉဃ ࢸႆᢋỆˤạ٭҄ửਦ೅ểẲềẟỦẇ

ဃࢸଐᱫ൑ƴࢽǒǕƨᨼӳ'252Ʊ25Ʒ Хນࣖሉࣱ

2 mV 2 ms

PS amplitude

0 2 4 6 8 10 12

0 100 200 300 400 500 600 700

PS amplitude (mV)

stimulation (PA) control 0 2 4 6 8 10

0 100 200 300 400 500 600 700

fEPSPslop (mV/ms)

stimulation (PA) control

PND13 PND14 PND15

PND16 PND17 PND18

2 ms

fEPSP slope

2 mV

•

ᵮᵬᵢᵏᵓẦỤᵮᵬᵢᵏᵔỆẦẬềཎỆᵮᵱਰࠢỉХນࣖሉࣱầɟൢỆʨᡶẴỦẇ

இᡈƷჷᙸᲴෙᬔ%#᪸؏ƴƓƚǔURKPG FGPUKV[ƷဃࢸفьƱƦƷϋ׆ࣱᅕኺ෇ѣ̔܍ࣱ

• ᵡᵟᵏ᪸؏ỆấẬỦᶑᶎᶇᶌᶃᴾᶂᶃᶌᶑᶇᶒᶗỊဃࢸᵏᵒଐᱫЭࢸỆ࣯ᡮỆفьẲஇٻ ểễỦẇ

• ᵩᶇᶐᵐᵌᵏửᢅйႆྵẰẶẆϋ׆ࣱᅕኺ෇ѣử৮СẲẺἰỸἋỂỊᶂᶃᶌᶑᶇᶒᶗỉ فьầᛐỜỤủẵẆẮỉᶂᶃᶌᶑᶇᶒᶗỉفьầϋ׆ࣱᅕኺ෇ѣỆ̔܍Ẳềẟ ỦẮểầᅆՐẰủỦẇ

ᵨᶍᶆᶌᶑᶍᶌᵋᵴᶃᶌᶉᵿᶒᶃᶑᶆ ᵣᵫ ᶃᶒᴾᵿᶊᵊᴾᵢᶃᶔᶃᶊᶍᶎᶋᶃᶌᶒ ᵐᵎᵏᵓ

ϋ׆ࣱƷᐻڠࣱ෇ѣǛᛦራƢǔ ßCODKGPVà)#$#

„ ḛᵟᶋᶀᶇᶃᶌᶒḜᴾᵥᵟᵠᵟểỊῒῒῒ

„ ἉἜἩἋ᧓ᨤẦỤ᡹ᏮẲềẨẺᅕኺˡᢋཋឋỉᶑᶎᶇᶊᶊᶍᶔᶃᶐᵆๆ්ᵇ

„ ἂἼỴኬᏘẦỤỉᵡᵿ ^ᵐᵉ ᩼̔܍ࣱᵍ᩼ݱᏘࣱỉἩἿἍἋỆợỦ્

Ј

„ ἚἻὅἋἯὊἑὊửʼẲẺᡞ᠞ᡛ ễỄử̓ዅเểẴỦᵥᵟᵠᵟỉẮểẇ

„ ẮỉḛᵿᶋᶀᶇᶃᶌᶒᴾᵥᵟᵠᵟḜầờẺỤẴờỉầḛᶒᶍᶌᶇᶁᴾᶇᶌᶆᶇᶀᶇᶒᶇᶍᶌḜểԠịủ Ủਤዓႎễ৮СࣱᛦራೞᏡỂẝụẆᅕኺׅែᐻڠࣱỉᛦራỆ᧙ ɨẲềẟỦểᎋảỤủềẟỦẇ

„ ᵮᵱਰࠢỆݣẲềᵿᶋᶀᶇᶃᶌᶒᴾᵥᵟᵠᵟỆợỦ৮СࣱᛦራầỄỉợạỆ᧙

ɨẲềẟỦẦẆộẺᏒဃ஖୵ᩧỆợụẮỉᛦራೞᏡầࢨ᪪ửӖẬ

ỦỉẦỄạẦử౨᚛ẴỦẮểỂᚸ̖ਦ೅ỆጟậỦẇ

)

)#$# _# Ӗܾ˳ਛ৴ᕤȓǯǯȪȳ$/+ŴŒ/

Ʒ25ਰࠢƴݣƢǔࢨ᪪

control

(+)BMI

2 mV 5 ms

(-)BMI ᵆᵉᵇᵠᵫᵧỆấẬỦᵮᵱਰࠢ

ᵠᵫᵧൔᵆᵠᵫᵧᴾᶐᵿᶒᶇᶍᵇ ᵆḗᵇᵠᵫᵧỆấẬỦᵮᵱਰࠢ

ᾌ

ХນࣖሉࣱỉʨᡶầЈྵ

ẴỦ଺஖ỉЭࢸỂൔ᠋

(-)B MI

(+)BMI 2

4 6 8 1 0

PS amplitude (mV)

0

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6

B M I ra tio

Probability

0 0

(-)B MI

(+)BMI 4

8 1 2 1 6 2 0 2 4

PS amplitude (mV)

0

0 .51 .01 .52 .02 .53 .03 .54 .0 0 .2

0 .4 0 .6

0 B M I ra tio

Probability

0

Control (PND14-15)

Control (PND16-17)

A

B

25ਰࠢƴݣƢǔ$/+ƷјௐᲴദࠝݣༀ፭Ʒئӳ

0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 0 . 2

0 . 4 0 . 6

B M I ra tio

P robabi lit y

0 0

P N D 1 4 -1 5 P N D 1 6 -1 7

Control

25ਰࠢƴƓƚǔ$/+ൔƷЎࠋ

• ᵮᵬᵢᵏᵒᵋᵏᵓỂỊᵠᵫᵧỆợụᵮᵱਰࠢ

ỉفࢍầᛐỜỤủỦ

ḵ ᵿᶋᶀᶇᶃᶌᶒᴾᵥᵟᵠᵟỆợỦ৮Сࣱ

ᛦራầ᧙ɨẲềẟỦẇ

• ᵮᵬᵢᵏᵔᵋᵏᵕỆễỦểᵠᵫᵧỉјௐỊỖ ểỮỄᛐỜỤủễẟẇ

ḵ ᵿᶋᶀᶇᶃᶌᶒᴾᵥᵟᵠᵟỆợỦ৮Сࣱ

ᛦራỉ᧙ɨầෞڂẲềẟỦ

• ХນࣖሉࣱƴݣƠƯCODKGPV )#$#ƴǑǔ ৮Сࣱᛦራƕ܍נƢǔŵ

• ƜƷᛦራೞᏡƕဃࢸႆᢋƴˤƍ٭҄ƢǔƜƱ ƕХນࣖሉࣱƕɟൢƴʨᡶƢǔƜƱƴ᧙ᡲƠ ƯƍǔᲹ

Ȑȫȗȭᣠ82#ȈȪȖȁȫǹǺ6$6 ƷᏒဃ஖୵ᩧƴǑǔࢨ᪪

¾ ׅែᐻڠࣱᾉᵮᵱਰࠢểᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃỆấẬỦХ ນࣖሉࣱửਦ೅ểẴỦᚸ̖

vaginal plugs/smears

GD 0 15

chemical administration Pregnant dams

21

Pups (in lactation period)

0 20

Hippocampal slice preparation

Stimulus-response relationship

13 14 15 16 17 18 PND

14 15 16 17 Effect of BMI on PS

PND Evaluation

PND

ᚸ̖ȗȭȈdzȸȫ

cal 21

ᵴᵮᵟίᵑᵎᵎᴾᶋᶅᵍᶉᶅᵇ ᵭᵰ ᵲᵠᵲᵆᵐᵎᴾᶋᶅᵍᶉᶅᵇ

ෙᬔǹȩǤǹ೅ஜ˺঺ƱᛔႆᩓˮƷਦ೅

2 ms fEPSP slope

2 mV

2 mV 2 ms PS amplitude PS

fEPSP

stim

ෙᬔ ᨺࣱἻἕἚ ᵆᵏᵑḗᵏᵖଐᱫᵇ

ෙᬔ

ᐻڠࣱἉἜἩἋỉࢍẰ ᵬᵿ

^ᵉ

ἓἵ἟Ἵỉ෇ࣱ҄

ểӷ஖ࣱ

ᚡ᥵ဇࣇݱᩓಊểХນ ᩓಊỉˮፗ᧙̞

ෙᬔ

੔ӕ ἋἻỶἋ

೅ஜ˺ᙌ

ᩓಊửᚨፗẴỦ

ҽẰ600 μm

ဃ

ဃࢸଐᱫ൑ƴࢽǒǕƨᨼӳ'252Ʊᨼӳǹȑ Ǥǯᩓˮ25ƷХນࣖሉࣱᲴ82#

2 mV 2 ms

PS amplitude

0 2 4 6 8 10 12

0 100 200 300 400 500 600 700

PS amplitude (mV)

stimulation (PA) control

0 2 4 6 8 10 12

0 100 200 300 400 500 600 700

PS amplitude (mV)

stimulation (PA) VPA 0

2 4 6 8 10

0 100 200 300 400 500 600 700

fEPSPslop (mV/ms)

stimulation (PA) control

PND13 PND14 PND15

PND16 PND17 PND18

0 2 4 6 8 10

0 100 200 300 400 500 600 700

fEPSPslope (mV/ms)

stimulation (PA) VPA

2 ms

fEPSP slope

2 mV

•

ݣༀ፭ỂỊᵮᵬᵢᵏᵓẦỤᵮᵬᵢᵏᵔỆẦẬềཎỆᵮᵱਰࠢỉХນࣖሉࣱầɟൢỆʨᡶẲẺẇ

•

ẲẦẲễầỤᵴᵮᵟ፭ỂỊᵮᵬᵢᵏᵒẦỤᵮᵬᵢᵏᵓỉ଺஖ỆẦẬềХນࣖሉࣱỉʨᡶầࢹẉỆЈ

ྵẲڼỜẺẇ

82#Ꮢဃ஖୵ᩧƴƓƚǔХນࣖሉࣱƷႆ

ᢋƴˤƏ٭҄

• Хນᩓ්ᵔᵎᵎ᷈ᵟỆấẬỦᵮᵱਰࠢểᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃửẆဃࢸᵏᵑଐ ᱫẦỤᵏᵖଐᱫỆỪẺỦ٭҄ểẲềϐࡇἂἻἧ҄ẲẆݣༀ፭ểൔ

᠋ẲẺẇ

• ᵴᵮᵟ፭ỉᵮᵱਰࠢỂỊဃࢸᵏᵒଐᱫểᵏᵓଐᱫỂẆᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃỂ ỊဃࢸᵏᵓଐᱫỂݣༀ፭ỆൔỔХນࣖሉࣱầஊॖỆفٻẲề ẟẺί ^ᵈ ᶎᵚᵎᵌᵎᵓᵊᴾ ^ᵈᵈ ᶎᵚᵎᵌᵎᵏὸẇ

0 2 4 6 8 10 12

12 13 14 15 16 17 18 19

PS amplitude (mV)

postnatal day

control 300 mg/kg

* **

VPA

0 2 4 6 8 10

12 13 14 15 16 17 18 19

fEPSPslope (mV/ms)

postnatal day

cont VPA300

**

control VPA

ဃࢸଐᱫ൑ƴࢽǒǕƨᨼӳ'252Ʊᨼӳǹȑ Ǥǯᩓˮ25ƷХນࣖሉࣱᲴ6$6

2 mV 2 ms

PS amplitude

0 2 4 6 8 10 12 14 16

0 100 200 300 400 500 600 700

PS amplitude (mV)

stimulation (μA) control

0 2 4 6 8 10 12 14 16

0 100 200 300 400 500 600 700

PS amplitude (mV)

stimulation (μA) TBT 0

2 4 6 8 10 12

0 100 200 300 400 500 600 700

fEPSPslope (mV/ms)

stimulation (PA)

PND13 PND14 PND15

PND16 PND17 PND18

control

0 2 4 6 8 10 12

0 100 200 300 400 500 600 700

fEPSPslope (mV/ms)

stimulation (PA) TBT

2 ms

fEPSP slope

2 mV

• ݣༀ፭ểൔ᠋ẲềẆᵲᵠᵲ፭ỂỊᵮᵬᵢᵏᵓẦỤᵮᵬᵢᵏᵔỆЈྵẴỦХນࣖ

ሉࣱỉʨᡶầݱẰẦẾẺẇ

6$6Ꮢဃ஖୵ᩧƴƓƚǔХນࣖሉࣱƷႆᢋ ƴˤƏ٭҄

0 2 4 6 8 10 12

12 13 14 15 16 17 18 19

f EPSP slope (mV/ms)

postnatal day control TBT 0

2 4 6 8 10 12 14 16 18

12 13 14 15 16 17 18 19

PS amplitude (mV)

postnatal day control

** TBT

• Хນᩓ්ᵔᵎᵎ᷈ᵟỆấẬỦᵮᵱਰࠢểᶄᵣᵮᵱᵮ ᶑᶊᶍᶎᶃửẆဃࢸᵏᵑ ଐᱫẦỤᵏᵖଐᱫỆỪẺỦ٭҄ểẲềϐࡇἂἻἧ҄ẲẆݣༀ፭ ểൔ᠋ẲẺẇ

• ᵲᵠᵲ፭ỂỊᵮᵱਰࠢỉ૾ỂဃࢸᵏᵔଐᱫỆấẬỦХນࣖሉࣱầ

˯ẪẆݣༀ፭ểൔỔềஊॖࠀầẝẾẺᵆ ^ᵈᵈ ᶎᵚᵎᵌᵎᵏᵇẇ

Ȑȫȗȭᣠ82#ȈȪȖȁȫǹǺ6$6 ƷᏒဃ஖୵ᩧƴǑǔࢨ᪪

¾ ׅែᐻڠࣱᵆᵮᵱਰࠢᵇỆݣẴỦ৮Сࣱᛦራỉဃ ࢸႆᢋửਦ೅ểẴỦᚸ̖

)#$# _# Ӗܾ˳ਛ৴ᕤȓǯǯȪȳ$/+ŴŒ/

Ʒ25ਰࠢƴݣƢǔࢨ᪪

control VPA

(+)BMI

2 mV 5 ms

(-)BMI

ᵆᵉᵇᵠᵫᵧỆấẬỦᵮᵱਰࠢ

ᵠᵫᵧൔᵆᵠᵫᵧᴾᶐᵿᶒᶇᶍᵇ

ᵆḗᵇᵠᵫᵧỆấẬỦᵮᵱਰࠢ

ᾌ

ХນࣖሉࣱỉʨᡶầЈྵ

ẴỦ଺஖ỉЭࢸỂൔ᠋

• ᵴᵮᵟᏒဃ஖୵ᩧᾉ

• ᵮᵬᵢᵏᵒᵋᵏᵓể ᵮᵬᵢᵏᵔᵋᵏᵕ

• ᵲᵠᵲᏒဃ஖୵ᩧᾉ

• ᵮᵬᵢᵏᵒểᵮᵬᵢᵏᵔ

(-)B MI

(+)BMI 2

4 6 8 1 0

PS amplitude (mV)

0

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6

B M I ra tio

Probability

0 0

(-)B MI

(+)BMI 4

8 1 2 1 6 2 0 2 4

PS amplitude (mV)

0

0 .51 .01 .52 .02 .53 .03 .54 .0 0 .2

0 .4 0 .6

0 B M I ra tio

Probability

0

Control (PND14-15)

Control (PND16-17)

A

B 2

25ਰࠢƴݣƢǔ$/+ƷјௐᲴദࠝݣༀ፭Ʒئӳ

(-)B MI

(+)BMI 0

4 8 1 2 1 6

PS amplitude (mV)

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6

0 B M I ra tio

Probability

0 (-)B

MI (+)BMI 0

2 4 6 8 1 0 1 2 1 4

PS amplitude (mV)

0 .51 .01 .52 .02 .53 .03 .54 .0 0 .2

0 .4 0 .6

B M I ra tio

Probability

00

VPA (PND14-15)

VPA (PND16-17)

25ਰࠢƴݣƢǔ$/+ƷјௐᲴ82#୵ᩧ፭Ʒئӳ A

B

0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 0 . 2

0 . 4 0 . 6

B M I ra tio

P robabi lit y

0 0

P N D 1 4 -1 5 P N D 1 6 -1 7

0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 0 . 2

0 . 4 0 . 6

B M I ra tio

P robabi lit y

0 0

P N D 1 4 -1 5 P N D 1 6 -1 7

Control VPA

25ਰࠢƴƓƚǔ$/+ൔƷЎࠋᲴ82#

• ݣༀ፭ỂᛐỜỤủỦႆᢋỆˤạЎࠋỉ٭҄Ịᵴᵮᵟ፭ỂᛐỜỤủễẟẇ

• ݣༀ፭ὉᵮᵬᵢᵏᵔᵋᵏᵕẦỤࢽỤủẺᵠᵫᵧൔỉЎࠋỊᵴᵮᵟ፭ὉᵮᵬᵢᵏᵔᵋᵏᵕẦỤࢽ ỤủẺЎࠋẻẬỂễẪᵮᵬᵢᵏᵒᵋᵏᵓẦỤࢽỤủẺЎࠋểỖỗɟᐲẲềẟỦẇ

#ODKGPV)#$#ƴǑǔ৮СࣱᛦራƷဃࢸႆᢋ ƴˤƏ٭҄ƕଔ஖ƴЈྵଔ༌ƠƯƍǔᲹ

25ਰࠢƴݣƢǔ$/+ƷјௐᲴ6$6ݣༀ፭Ʒئӳ

(-)B MI

(+)BMI 2

4 6 8 1 0

PS amplitude (mV)

0

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6 0 .8

B M I ra tio

Probability

00

(-)B MI

(+)BMI 0

4 8 1 2 1 6 2 0 2 4

PS amplitude (mV)

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6 0 .8

B M I ra tio

Probability

0 0

Control (PND14)

Control (PND16)

A

B

25ਰࠢƴݣƢǔ$/+ƷјௐᲴ6$6୵ᩧ፭Ʒئӳ

(-)B MI

(+)BMI 0

2 4 6 8 1 0

PS amplitude (mV)

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6 0 .8

B M I ra tio

Probability

0 0

(-)B MI

(+)BMI 0

5 1 0 1 5 2 0

PS amplitude (mV)

0 .5 1 .0

1 .5 2 .0

2 .5 3 .0

3 .5 4 .0 0 .2

0 .4 0 .6 0 .8

B M I ra tio

Probability

0 0

TBT (PND14)

TBT (PND16)

A

B

P N D 1 6 ( c o n t r o l) P N D 1 4 ( c o n t r o l)

0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 0 . 2

0 . 4 0 . 6 0 . 8

B M I ra tio

P robabi lit y

0 0

P N D 1 6 P N D 1 4

25ਰࠢƴƓƚǔ$/+ൔƷЎࠋᲴ6$6

0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 0 . 2

0 . 4 0 . 6 0 . 8

B M I ra tio

P robabi lit y

0 0

P N D 1 6 P N D 1 4

Control TBT

• ႆᢋỆˤạᵠᵫᵧൔЎࠋỉ٭҄Ịẟẵủỉ፭ỂờᛐỜỤủỦẇ

• ᵲᵠᵲ፭ὉᵮᵬᵢᵏᵔỉЎࠋỊݣༀ፭ᵍᵲᵠᵲ፭ὉᵮᵬᵢᵏᵒỉЎࠋểݣༀ፭Ὁᵮᵬᵢᵏᵔ ỉЎࠋểỉ᧓ỆˮፗẴỦẇ

#ODKGPV)#$#ƴǑǔ৮СࣱᛦራƷဃࢸႆᢋ

ƴˤƏ٭҄ƕͣ๛᡿ࡨƠƯƍǔᲹ

LJ LJƱNJ

„ ᵴᵮᵟᏒဃ஖୵ᩧἻἕἚỂỊׅែᐻڠࣱẆấợỎẮủỆݣẴỦ৮С

ࣱᛦራỉဃࢸႆᢋỆấẬỦଔ༌҄

„ ᵲᵠᵲᏒဃ஖୵ᩧἻἕἚỂỊׅែᐻڠࣱẆấợỎẮủỆݣẴỦ৮С

ࣱᛦራỉဃࢸႆᢋỆấẬỦ᡿ࡨᵆͣ๛ᾎᵇ ửᙸЈẲẺẇ

੉ʐ஖ƷෙᬔᅕኺׅែೞᏡƷႆᢋǛᚸ̖ƢǔƜƱƕŴႆᢋ

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ǔӧᏡࣱƕᎋƑǒǕƨŵ

2016 Autumn

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Contents lists available atScienceDirect

Neuroscience and Biobehavioral Reviews

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u b i o r e v

Review article

Autism spectrum disorder and attention-deficit/hyperactivity disorder in early childhood: A review of unique and shared characteristics and developmental antecedents

Janne C. Vissera,∗, Nanda N.J. Rommelsea,b, Corina U. Grevena,c,d, Jan K. Buitelaara,c aKarakter Child and Adolescent Psychiatry University Center, Nijmegen, The Netherlands

bRadboud University Medical Center, Donders Institute for Brain, Cognition and Behavior, Department of Psychiatry, Nijmegen, The Netherlands cRadboud University Medical Center, Donders Institute for Brain, Cognition and Behavior, Department of Cognitive Neuroscience, Nijmegen, The Netherlands

dKing’s College London, Medical Research Council Social, Genetic & Developmental Psychiatry Centre, Institute of Psychiatry, Psychology & Neuroscience, London, UK

Table 1

Summary of findings on temperament in children with (traits of) ASD or ADHD.

Temperament dimension 6–11 months 1–2 years 2–3 years 3–4 years 4–5 years

ASD ADHD ASD ADHD ASD ADHD ASD ADHD ASD ADHD

Approach/surgency

Composite score ↑^2,3*b ↓² ↓^2,3*ns12 ↓^3* ↑⁹ ↑⁹

Activity ↓^1,3*,5 ↑^7,8 ↓^3* ↑⁸ ↑^1,4 ↑⁸ ↑⁶↓^12e ↑⁶↓^12e

Perceptual sensitivity ↑² ns7 ↑² ns7 ns7

High intensity pleasure ↓² ↑¹⁰ ↑¹⁰

Positive affect ↓² ↓⁴

Positive anticipation/ non-shyness^a ↓^4c ↓⁶

Impulsivity ↓^12e ↑¹⁰ ↓^12e ↑¹⁰

Negative affect

Composite score ns3* ns3* ns3*↑¹² ns3* ↑^9,11 ↑⁹

Sadness/shyness^afear ↑¹ ns⁷ ns⁷ ↑^1,2,4ns¹² ↑⁸ns⁷ ↑¹² ↑¹²↑¹²

Anger ↑⁷ ↑⁸ ↑⁴ ↑⁸

Distress/discomfort reactions ↑¹³ ↑⁵ ↑¹²

Effortful control

Composite score ↓² ↓²ns¹² ns¹² ↓^9,10 ↓^9,10

Persistence/non-distractibility Vigilance/interest (ADHD) ↑1ns3* ↓7d,8 ns3* ↓8 ↑1ns3* ↓8

Cuddliness ↓² ↓²

Low intensity pleasure ↓2

Attention shifting ↓⁸ ↓⁵ ↓⁸ ↓^4,5 ↓¹² ↓¹²

Control of attention ↓⁴ ↓⁸

Inhibitory control ↓⁵ ↓^8,14 ↓^12e ↓¹³ ↓^12e

Fig. 1.Temperament traits in ASD and ADHD: ASD and ADHD share high levels of negative affect, but the underlying motivational and behavioral tendencies seem to differ, i.e. withdrawal vs approach in ASD vs ADHD, respectively. ASD and ADHD also share difficulties with control and shifting, but partly opposite behaviors seem to be involved, i.e. high persistence and low distractibility in ASD and poor sustained attention and high distractibility in ADHD.

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䠑ᅇ⬊≧ወ⫾䜢⧞䜚㏉䛧䛯ᏙⓎ౛䠊 ᾏእ䛾ᐙ᪘ᛶ⑕౛䛷䛿䚸

•

඲⬊≧ወ⫾䛸ྠ୍䛾⑓⌮ᡤぢ䠊

•

᰾ᆺ䛿ṇᖖ஧ಸయ䠊

• DNA䝯䝏䝹໬␗ᖖ䜢క䛖.

䠄Nature 2002䠅

• NALP7 㑇ఏᏊ䚸c6orf221 㑇ఏᏊኚ␗䛸㛵

㐃䛩䜛.䠄NatGenet 2006, Am J Hum Genet 2011䠅

•

㠀཯᚟౛䛷䜒ᐙ᪘Ṕ䜢᭷䛩䜛ᝈ⪅䛻䛿

NALP7 㑇ఏᏊኚ␗䛜ぢฟ䛥䜜䛶䛔䜛䠊

ẕ

∗

∗∗ 䝩䝰ኚ␗ẕ䛾

༸Ꮚ䛾䝯䝏䝹໬␗ᖖ䠛

ཷ⢭ᚋ䛾 䝯䝏䝹໬⥔ᣢ␗ᖖ䠛

ẕ

•

䜰䜲䝇䝷䞁䝗䠓䠔ᐙ⣔䛾඲䝀䝜䝮㓄ิゎᯒ⤖ᯝ䠊

•

୍ୡ௦䛷ᖹᆒ⣙䠒䠌ಶ䛾᪂つኚ␗䠊

•

∗ぶ䛾ຍ㱋䛸䛸䜒䛻䚸Ꮚ䛾᪂つኚ␗ᩘ䛿ቑ䛘䜛䠄∗䠎䠌ṓ䛷䠍䠑ಶ䚸∗䠐䠌ṓ䛷䠒䠑ಶ䠅䠊

•

ẕ䛿䚸䛒䜎䜚ຍ㱋䛻䜘䜛ኚ໬䛿䛺䛔䠄䛚䜘䛭䠍䠑ಶ䛟䜙䛔䠅䠊

•

㞝⏕Ṫ⣽⬊䛿䚸⏕ᾭ䛻䜟䛯䜚య⣽⬊ศ⿣䛩䜛䛯䜑䚸䜶䝷䞊䛜⵳✚䛩䜛䠊

Ꮚ䜈䛾ᙳ㡪䛿䚸ẕ⏤᮶䛾ᙳ㡪䠄⫾ඣᮇ䛾ẕయᰤ㣴≧ែ䜔ຍ㱋䠅䛰䛡䛷䛺䛟䚸

∗䛾ᰤ㣴≧ែ䜔ຍ㱋䜒䚸䜶䝢䝀䝜䝮䞉䝀䝜䝮཮᪉䛾ᶵᵓ䜢㏻䛨䛶స⏝䛩䜛䠊

ຍ㱋䛾ᙳ㡪䛿ẕ䛰䛡䛷䛿䛺䛔

Nature (2012) 488, 471-

475

ᐙ⣔䝃䞁䝥䝹䛷䛾ゎᯒ䛜㔜せ

㏆ぶᗘ䠍ᗘ

㑇ఏ᝟ሗ1/2ඹ᭷

㏆ぶᗘ䠍ᗘ

㑇ఏ᝟ሗ1/2ඹ᭷

㏆ぶᗘ䠍ᗘ

㑇ఏ᝟ሗ1/2ඹ᭷

⤌ྜ䜟䛫1/4୍⮴

ㄡ䜢ゎᯒ䛧䛶䜒20୓䛛ᡤ 㓄ิኚ໬ೃ⿵

⛥䛺㓄ิኚ໬

1,000⛬ᗘೃ⿵

୧ぶ䛸ẚ㍑䛷䛝䜜䜀100ಶᮍ‶

඗ᘵጜጒ᝟ሗ䛜䛒䜜䜀ᩘಶࠥᩘ༑ಶ ᐙ᪘᝟ሗ䛒䜚

ㄡ䜢ゎᯒ䛧䛶䜒20୓䛛ᡤ 㓄ิኚ໬ೃ⿵

ᐙ᪘᝟ሗ䛺䛧

⛥䛺㓄ิኚ໬

1,000⛬ᗘೃ⿵

᪤▱䛾⑌ᝈኚ␗䛜 ぢ䛴䛛䜙䛺䛔㝈䜚

⤠䜚㎸䜏䛿୙ྍ⬟

http://nrichd.ncchd.go.jp/irud-p/

IRUD- P@ncchd go jp

ᡂ⫱㻵㻾㼁㻰㻙㻼஦ົᒁ䠖䠌䠏䠉䠑䠐䠕䠐䠉䠔䠍䠏䠓

៞᠕㻵㻾㼁㻰㻙㻼஦ົᒁ䠖䠌䠏䠉䠑䠏䠒䠏䠉䠏䠕䠌䠒

ँ ः

ै ␷ ऒ

ᕼᑡ䞉ᮍデ᩿⑌ᝈ䜲䝙䝅䜰䝏䝤

ᅜ❧ᡂ⫱་⒪◊✲䝉䞁䝍䞊䞉䝞䜲䜸䝞䞁䜽

䞉 ⮫ᗋ᝟ሗ䜢క䛖⣙㻣㻘㻜㻜㻜⑕౛䛾᪤Ꮡ᳨య 䞉 ㇏ᐩ䛺࿘⏘ᮇ䞉ᑠඣᕼᑡ⑌ᝈ 䞉 ⫾ඣᮇ䞉ฟ⏕᫬䛾ṇ☜䛺⮫ᗋ᝟ሗ䛸ヨᩱ

䛂⩦័ὶ⏘≉␗ⓗ䛃䛸䛧䛶ሗ࿌

䛥䜜䛯㻯㻺㼂㻝㻣㡿ᇦ䛾䛖䛱㻤䛛 ᡤ䛿䚸ᡃ䚻䛾⊂⮬䝕䞊䝍᪥ᮏ

ேṇᖖศፔṔ㞟ᅋ䛷䜒ほᐹ䛥 䜜䛯䛣䛸䛛䜙䚸䛚䛭䜙䛟ὶ⏘䛸 䛿㛵ಀ䛾䛺䛔㻯㻺㼂䛸⪃䛘䜙䜜 䛯䠊

Human Reproduction vol 25, 2010

᪥ᮏே䛂ṇ ṇᖖ䛃ศፔ㞟ᅋ䝕䞊䝍䛾᭷⏝ᛶ

䜽䝻䝬䝏䞁䛸䝠䝇䝖䞁ಟ㣭

䜽䝻䝬䝏䞁

䝁䜰䝠䝇䝖䞁ඵ㔞య

DNA䛜ᕳ䛝䛴䛟

䝠䝇䝖䞁䝔䜲䝹

(䝯䝏䝹໬䞉䜰䝉䝏䝹໬

䛺䛹䛾ಟ㣭䜢ཷ䛡䜛)

䝠䝇䝖䞁䝯䝏䝹໬

䝠䝇䝖䞁䜰䝉䝏䝹໬

DNA䝯䝏䝹໬

䝚䜽䝺䜸䝋䞊䝮

䝦䝔䝻䜽䝻䝬䝏䞁

(㌿෗ᢚไ)

ᰁⰍయ

䝴䞊䜽䝻䝬䝏䞁

(㌿

㌿෗άᛶஹ㐍)

ᅜ

ᅜ㝿㐃ᦠ䛻䜘䜛䜶䝢䝀䝜䝮䝸䝣䜯䝺䞁䝇䝕䞊䝍ᩚഛ䛾ヨ䜏

• 90%௨ୖ䛻⣧໬䛧䛯ᶆⓗ⣽⬊

• DNA䝯䝏䝻䞊䝮ゎᯒ

• 6✀䛾䝠䝇䝖䞁ಟ㣭ゎᯒ

䠄H3K4me3, H3K4me1, H3K27me3, H3K9me3, H3K36me3,

H3K27ac䠅

• 䝖䝷䞁䝇䜽䝸䝥䝖䞊䝮 small RNA