! ! ! !Characterization of the mice deficient in G-substrate, a PKG substrate. PKG 基質G-substrate欠損マウスの特徴付け!
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Shogo Endo1, Fumihiro Shutoh2, Tung L. Dinh3, Takehito Okamoto2, Soichi Nagao2, and Masao Ito3!
!1Tokyo Metropolitan Institute of Gerontology, !
!2Lab for Motor Learning Control and 3Lab for Memory and Learning, RIKEN Brain Science Institute.!

遠藤昌吾1、首藤文洋2、Tung L. Dinh2、岡本武人2、!
永雄総一2、伊藤正男2!
1東京都老人総合研究所、2理研脳センター!

Introduction
NO(nitric oxide)-cGMP-PKG(cGMP-dependent protein kinase) pathway plays important role in the central nervous system.
However, the restricted distribution of the pathway components and the lack of information on the substrate for the PKG havehindered research to examine the physiological roles of the cGMP pathway.
We have characterized G-substrate as an excellent PKG substrate and as a potent protein phosphatase inhibitor localized incerebellar Purkinje cells. Further, we characterized the structure of mouse G-substrate gene and generated the G-substrategene-deficient mice. The homozygote G-substrate knockout (KO) mice didn’t show ataxia nor morphological abnormality in
cerebellar cells. However, the homozygote mice showed reduced cerebellar LTD (long-term depression) temporarily aroundpostnatal week (PW) 6 compared with wild-type (WT) mice. General behaviors of the mice are normal, however, the long-term
adaptation optokinetic response (OKR) of eye movement was impaired without any affect on short-term adaptation of OKR. はじめに�� � NO(一酸化窒素)-cGMP-PKG(cGMP-依存性 kinase) 系は中枢神経系で重要な役割を果たしている。しかし、PKG基質の未同定、PKGが限られた細胞に発現しているなどの理由から、cGMP系の生理機能解析はcAMPに比較しておくれている。
� 我々は小脳プルキンエ細胞に存在するG-substrateを単離し、強力なタンパク質ホスファターゼ阻害機能を明らかにした。さらに、G-substrate遺伝子欠損マウスを作出して、その生理機能をさらに解析した。遺伝子欠損マウス（-/-）では小脳やプルキンエ細胞等に明らかな形態変化は観察されなかったが、発達段階で一時的に小脳LTDが減弱していた。さらに、小脳依存性学習である視機性眼球応答(OKR)の短期順応は正常であったが、長期順応が著しく障害されていた。NO系が特異的に長期記憶に関わる可能性を示唆している。�
Behavioral examinations	

Histochemical examinations	

b12

Day
3a Gain=b/a45

I hrtraining

Generation of G-substrate KO mice.
(A) Mouse G-substrate gene structure and targeting
vector for the generation of G-substrate KO mice.
(B) Restriction-enzyme-digested genomic DNA wassubjected to Southern blot hybridization analysis. Theband shift due to the homologous recombination wasconfirmed by probing the blot with 32P-labeled 3’- and5’-probes indicated in the figure. The removal of theselection marker, pgk-Neo cassette, was also
confirmed by Southern blot analysis using the 32Plabeled 3’-probe.	

Cerebellar structures of WT and KO mice.
(A)Nissl staining of cerebellar slices obtained from WT and KO
mice. The thin sections (30 µm) were obtained fromparaformaldehyde-fixed brain using a cryostat and were subjected
to Nissl staining.
(B) HE (hematoxylin and eosin) staining of brain slices obtained
from WT and KO mice.
(C) Fluorescence images of slice obtained from G-substrate KO
mice. The frozen thin sections (20 µm) were observed under afluorescence microscope. AFAP, a GFP derivative, was expressed
under the control of the G-substrate promoter.	

Electrophysiological examinations	Comparison of basic electrophysiological properties of Purkinje cells in the G-substrategene knockout and wild-type mice.*

Age-dependent expression ofcerebellar LTD in slice preparationfrom G-substrate KO mice.
(A) Averaged time profile of 3groups of WT Purkinje cells, shownwith different colors. Ordinate,rising slope of PF-EPSPs relative
to the average measured 5 min
before conjunction. The shadedband indicates the application ofconjunction of PF stimulation anddepolarizing pulses. In brackets,
number of Purkinje cells tested.
Vertical bars, standard errors.
(B) Similar to (A) but for Gsubstrate-deficient Purkinje cells.
(C) Histograms showing LTD
amplitude at 41-50 min for different
PW (postnatal week) in WT and Gsubstrate-deficient Purkinje cells.
Filled columns are G-substrate KO
mice, and empty columns are WT
littermates. Vertical bars, standarderrors.
The numbers of cells used for theexperiments are as follows; for WT
mice PW4 (12), PW5 (23), PW6 (14),PW7 (10), PW8 (14), PW10-15 (23);for KO mice PW4 (10), PW5 (9),PW6 (9), PW7 (6), PW8 (11),PW10-15 (29).	

Mice lines
Electrophysiologicalproperties
Membrane potential(mV)

AgePW4
PW5
PW6
PW7
PW8
PW10-15Knockoutmean ± SE (n)-70.1 ± 1.9 (10)-62.7 ± 1.3 (22)-60.3 ±1.4 (13)-59.8 ± 1.3 (9)-59.8 ± 0.4 (11)-60.3 ± 0.6 (56)Wild-typemean ± SE (n)-72.9 ± 0.5 (7)-62.7 ± 0.8 (22)-59.9 ± 1.0 (17)-61.6 ± 0.8 (21)-61.0 ± 1.4 (19)-60.9 ± 1.8 (16)Membrane resistance
(M!)

PW4
PW5
PW6
PW7
PW8
PW10-15

33.8 ± 0.8 (8)33.7 ± 0.5 (20)33.2 ± 0.9 (12)30.3 ± 1.0 (9)30.1 ± 0.7 (13)30.1 ± 0.4 (59)33.1 ± 0.3 (9)33.4 ± 0.7 (22)30.0 ± 0.9 (17)30.6 ± 0.8 (21)30.1 ± 0.8 (19)31.5 ± 0.9 (16)PF-EPSP peak size
(mV)

PW4
PW5
PW6
PW7
PW8
PW10-158.8 ± 0.3 (10)8.6 ± 0.3 (23)8.1± 0.3 (13)8.3 ± 0.8 (9)8.1 ± 0.7 (14)8.1 ± 0.2 (57)8.3 ± 0.3 (10)8.4 ± 0.3 (24)8.9 ± 0.4 (19)8.6 ± 0.4 (21)8.2 ± 0.5 (19)8.4 ± 0.2 (16)PF-EPSP rise time
(10-90%) (msec)PW4PW5PW6PW7PW8PW10-15

5.5 ± 0.5 (10)5.9 ± 0.3 (23)6.0 ± 0.3 (14)4.1 ± 0.3 (9)4.3 ± 2.4 (14)4.4 ± 0.1 (37)5.6 ± 0.4 (10)5.1 ± 0.2 (24)4.6 ± 0.2 (19)4.2 ± 0.3 (21)5.0 ± 0.3 (19)4.6 ± 0.3 (16)PF-EPSP half width(msec)

PW4
PW5
PW6
PW7
PW8
PW10-15

26.3 ± 1.4 (10)28.6 ± 0.8 (23)28.3 ± 1.4 (14)24.6 ± 1.4 (9)23.7 ± 1.0 (14)24.0 ± 0.5 (57)25.5 ± 0.9 (10)25.2 ± 0.6 (24)28.5 ± 1.2 (19)23.5 ± 0.8 (21)25.4 ± 1.0 (19)24.3 ± 0.8 (16)PF-EPSP
paired-pulse
facilitation (before/afterconjunction)

PW4

1.34 ± 0.04(10)/1.31 ± 0.04(10)1.31 ± 0.04(13)/1.30 ± 0.04(11)1.37 ± 0.06(7)/1.34 ± 0.07 (8)1.34 ± 0.02(44)/1.31 ± 0.02(35)1.39 ± 0.02 (34)/
1.41 ± 0.02 (28)1.42 ± 0.06 (9)/1.42 ± 0.09 (8)1.46 ± 0.07 (5)/1.46 ± 0.09 (5)1.42 ± 0.03 (9)/1.35 ± 0.01 (8)1.39 ± 0.03 (32)/1.35 ± 0.02 (22)1.36 ± 0.04 (9)/1.31 ± 0.05 (6)86.1± 1.6 (5)83.5± 3.3 (4)88.1 ± 9.0 (4)81.8 ± 4.3 (4)80.0 ± 1.4 (10)

PW5
PW6
PW7
PW10-15

Climbing fiber responses
Initial peak (mV)
Duration (msec)

PW6+7
PW10-15
PW6+7
PW10-1580.0 ± 4.1 (10)*These data were analyzed statistically with factorial ANOVA. Genotype-dependent
differences were observed only in paired pulse facilitation, which was slightly smaller in G
substrate-deficient Purkinje cells than wild-type both before (P=0.017) and after (P=0.042)
conjunction, but no other significant difference was observed in any of other listed items.
Age-dependent variations were observed in membrane potential, membrane resistance,
PF-EPSP rise time, and PF-EPSP half width (P<0.001), but not in others (P>0.05). The
observed differences, however, were rather small, and their implications in LTD induction
or OKR adaptation are unclear. CS, conjunctive stimulation.

OKR dynamics of WT and G-substrate KO mice.
Left - Schematic diagram of OKR measurements. Eye movement inducedby oscillating screen was observed through CCD camera. The gain is
calculated maximum eye movement (b) divided by screen movement (a).
Right - Mice (12 week old) were subjected to sinusoidal screen oscillation(15° peak-to-peak, 0.11-0.33 Hz) at a maximum velocity of 1.7-10.5 °/s.
(A)Screen velocity-dependency of OKR gains.
(B) OKR phases. Positive and negative values respectively indicate phasedelay and advance. Open columns are WT mice and filled columns areKO mice. 	
Eyeblink conditioning.	
Performance was expressedas an index of CR% (thefrequency of the conditioned
response in the valid trials).	
(A) The WT mice (n=11) andKO mice (n=10) were trainedin a delay paradigm. In thisparadigm, the conditioned
stimuli preceded and coterminated with theunconditioned stimuli.		
(B) The WT (n=8) and KO mice(n=8) were trained in a trace
paradigm.
In the trace
paradigm, a stimulus-freetrace interval of 500 ms wasinterposed between the CS
and the US.
Filled circles, WT mice; opencircles, KO mice. 		

Long-term OKR adaptation.
WT (n=7) and KO mice (n=8) at PW12-15 were exposed to
1-hr sustained screen oscillation every day for 5 days.
The mice were kept in the dark except during the screen
oscillation. (A)Daily OKR adaptation. ∆Gain, gain changes
obtained after 1-hr oscillation on each day. Filled columns
are KO mice, and empty columns are WT littermates.
(B) Cumulative OKR gain change induced through 5-daysession. Ordinate, ∆Gain increases from the start gains
on each day as measured from the start gain on day 1.
Open circles are for WT and filled circles for KO mice. 		
Locomotion test- Rotor rod andtreadmill.
(A)The rotor rod test was carriedout using WT (n=5, blue) and KO(n=6, red) mice at 8 rpm. Falllatency was measured.
(B) Representative stick figures of
one complete step cycle (leftpanel, stance; right panel, swing)of a right hindlimb on the treadmill
at a belt speed of 8 m/min.
(C) The joint angles of the knee
and the ankle in sagittal plane areshown during a normalized step
cycle in WT (blue, n=5) and KOmice (red, n=5). The patterns ofthe joint angular movements weresimilar in WT and KO mice. Bothjoints in KO mice were maintainedin a slightly more extended
position compared to those in theWT mice. 	

Conclusions
1) The G-substrate KO mice did not express neither mRNA nor protein of G-substrate and they survived and mate normally.
2) The G-substrate KO mice did not show ataxia and no significant difference was observed between WT and G-substrate KO mice inrotorod test and other general behavior analyses.
3) The temporal reduction of cerebellar LTD around 6-week old G-substrate KO mice may suggest the phosphtase mechanisms
underlying cerebellar LTD might undergo the molecular change during development (See Launey et al, PNAS, 2004).
4) Slow long-term OKR adaptation in G-substrate KO mice may indicate the involvement of G-substrate in OKR consolidation.
5) The mice deficient in PKG also showed the impaired long-term adaptation in VOR without any effect on short-term one (Feil et al,2003). NO pathway may be involved specifically in long-term memory in eye movement.
For further info, please refer to Endo et al, PNAS 106, 3525-3530, 2009.