Neural circuits for eyeblink conditioning in rabbitshas been studied in detail, however, the basicknowledge on eyeblink conditioning in mice remainslimited. In the present study, we examined the role ofGABAA receptors in the mice deep cerebellar nuclei(DCN) for delay eyeblink conditioning. Bilateralinjection of Muscimol (MSC) and Picrotoxin (PTX)significantly impaired learned eyeblink responses(LER) in an acquisition test. MSC-injected mice couldnot acquire LER, however, PTX-injected may haveacquired LER, suggesting the distinctive effect ofthese drugs in DCN. Bilateral injection of MSC and PTXalso impaired the retention of acquired LER in a 7-dayretention test. Furthermore, ipsilateral injections ofMSC and PTX impaired the acquired LER as much asbilateral injection of them. Contralateral MSCinjections also impaired the expression of LER, butcontralateral PTX injections only partially impairedeyeblink conditioning. These results reveal thatGABAA receptors in DCN play essential roles in boththe acquisition and the expression of mouse eyeblinkconditioning.

1 2 3 4 5

Habituation (2 days)

Eyeblink training (7 days)

MSC, PTX, aCSF injection

days10 11 12 13 14 15 16

Retention test (7 days)

(2) Retention test

MSC and PTX impaired performance of rotor rod tests

Results

Introduction

　

Delay conditioning paradigm

CS

US

600 ms, 10kHz, 61dB

100 ms

ITI: 20-40 sec50 trials / day

Animals: Male C57BL/6 mice 8–9 weeks old.

Surgery:(1) Drug injection into the cerebellar nucleibilaterally (AP = -6.05, ML = ±1.80, DV = -2.25 mm).(2) Electromyogram (EMG) recording for eyeblinkresponses; Stainless wires were implanted underthe left upper eyelid.

Drug injection: 0.2 µl of 10 mM muscimol,200µM picrotoxin, aCSF (artificial cerebrospinal fluid )(0.15 µl / min)

Methods

Tips of internal cannulae were positioned in the cerebellar nuclei

(4) Histology

(1) Acquisition test

　1 2 3 4 5 6 7 8 9

Habituation (2 days) Eyeblink training (11 days)

MSC, PTX, aCSF injection aCSF (for 4 days)

days10 11 12 13

Conclusions

　Abstract

(5) Rotor-rod test

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day1

aCSF (n=7)Muscimol (n=7)Picrotoxin (n=5)

day1 day2 day 4 day 5 day 8 day 9

Fall

late

ncy

(Sec

)

Sessions

Tests after injection 1. Bilateral Muscimol-injected mice could not acquire learned eyeblink responses, however, Picrotoxin-injected mice may have acquired them.

2. Bilateral injection of Muscimol and Picrotoxin impaired the retention of acquired learned eyeblink responses.

3. Both ipsilateral and contralateral injection of of Muscimol and Picrotoxin impaired expression of learned eyeblink responses.

GABAA receptors play roles in acquisition and retention ofmouse eyeblink conditioning in the cerebellar nuclei

T. Sakamoto, T. Arasaki, & S. Endo （坂本敏郎 ・ 新崎智子 ・ 遠藤昌吾）Unit for Molecular Neurobiology of Learning & Memory （記憶と学習の分子神経生物学ユニット）Okinawa Institute of Science and Technology （沖縄科学技術研究基盤整備機構）

（小脳核GABA受容体はマウス瞬目反射条件づけの獲得と保持に重要な役割を持つ。）

Unilateral injection of MSC and PTX alsoimpaired retention of eyeblink learning

(3) Laterality test

　

Habituation (2 days)

Eyeblink training (7 days)

Bilaterally

days

Laterality test (12 days)

Ipsilaterally Contralaterally

3 4 5 6 7 8 9　10 11 12 13 14 15 16　17 18 19 20 211 26 7 8 9　

MSC, PTX, aCSF injection

-5.88

-6.00

PicrotoxinaCSF Muscimol

-6.12

-6.24

-6.36

aCSF Picrotoxin Muscimol

0

10

20

30

40

MuscimolPicrotoxinaCSF

Cell

Num

bers

(6) Numbers of cells around injection sites

No significant differences in the average number of Hu-immunopositiveneurons in MSC-, PTX-, and aCSF-injected mice

　 Inferior oliveCerebellar nuclei

Pontine nuclei

Tone CSEyeblink CR

Shock US

Red nucleus

Climbingfiber

Mossy fiber

Purkinje cell

Neural circuit for eyeblink conditioning

Basic knowledge for neural circuits on mouse eyeblink conditioning remains limited.

The role of bilateral and unilateral deep cerebellarnuclei in acquisition and retention of mouseeyeblink conditioning was examined.

GABAA receptors in cerebellar nuclei play essential roles in both the acquisition and the expression of mouse eyeblink conditioning.

Summary

Please contact Toshiro Sakamoto e-mail adress: sakamoto@oist.jp

Injection of MSC and PTX impaired retention of eyeblink learning

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LER = CR - SERLER (Learned Eyeblink Response) CR (Conditioned Response) SER (Spontaneous Eyeblink Response

Picrotoxin

Muscimol

aCSF

200 µm100 µm