Social isolation anxiety induced synaptic failure restored by dihydromyricetin

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Content SOCIAL ISOLATION ANXIETY INDUCED SYNAPTIC FAILURE RESTORED BY
DIHYDROMYRICETIN
B y
Ji fe ng Z ha ng
A T he si s Pre se nt e d t o t he
FA C UL T Y OF T HE USC SC HOOL OF PHAR MAC Y
UNIVE R SIT Y OF SOUT HE R N C AL IFOR NIA
In Pa rt i a l Ful fi l l m e nt of t he
R e qui re m e nt s for t he De gre e
MAST E R OF SC IE NC E
C l i ni c a l a nd E xpe ri m e nt a l T he ra pe ut i c s
August 2021
C opyri ght 2021 Ji fe ng Z ha ng
i i
A c k n o w l e d g e m e n t s
T i m e f l i e s s o f a s t a nd i t i s unbe l i e va bl e t ha t I w i l l gr a dua t e f r om U ni ve r s i t y of S out he r n
C a l i f or ni a S c hool of P ha r m a c y a s a m a s t e r s t ude nt i n C l i ni c a l a nd E xpe r i m e nt a l T he r a pe ut i c
( C X P T ) m a j or . B e c a us e of t he C O V I D - 19, e ve r yone e nc ount e r s va r i ous di f f i c ul t i e s i n e i t he r hi s
( he r ) l i f e , w or k or s t udy dur i ng t hi s s pe c i a l pe r i od. F i r s t l y, I w a nt t o e xpr e s s m y gr a t i t ude t o m y
m e nt or P r of . J i ng L i a ng f or he r c ons t r uc t i ve s ugge s t i ons on m y e xpe r i m e nt a nd t he s i s . S e c ondl y,
I w a nt t o s a y t ha nk you t o t he P hD s t ude nt A l z a hr a w ho gui de s m e da i l y e xpe r i m e nt s a nd c om e s
up w i t h s om e ne w i de a s a nd a l l ot he r m e m be r s i n P r of . D a r yl D a vi e s l a b. O f a l l t he m e m be r s , I
w oul d m os t l i ke t o t ha nk C he n a nd R a y f or t he i r he l p not onl y i n s t udy but a l s o i n l i f e . I a m a l s o
de e pl y i nde bt e d t o ot he r t w o c om m i t t e e m e m be r s - - P r of . D a r yl D a vi e s a nd P r of . P a ul B e r i nge r .
F i na l l y, I w oul d l i ke t o t ha nk m y pa r e nt s f or s uppor t i ng m y de c i s i on t o s t udy i n t he U S A .
T ha nks t o a l l t he pr of e s s or s , s t ude nt s a nd s t a f f of t he S c hool of P ha r m a c y i n U S C f or t he i r c a r e
a nd he l p dur i ng t he s e t w o ye a r s . F i ght on!
i i i
T A B L E O F C O N T E N T S
A c know l e dge m e nt s ..........................................................................................................................i i
L i s t of F i gur e s .................................................................................................................................i v
A bbr e vi a t i ons ...................................................................................................................................v
A bs t r a c t ...........................................................................................................................................vi
I nt r oduc t i on......................................................................................................................................1
C ha pt e r 1: M a t e r i a l s a nd m e t hods ..................................................................................................5
A ni m a l s ubj e c t s a nd s oc i a l i s ol a t i on m ode l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
D r ug pr e pa r a t i on a nd a dm i ni s t r a t i on.............................................................................6
B e ha vi or a l t e s t ...............................................................................................................7
W e s t e r n bl ot s .................................................................................................................8
B D N F E L I S A ..............................................................................................................10
H ydr oge n pe r oxi de a s s a y.............................................................................................11
B r a i n T i s s ue P r e pa r a t i on a nd I m m unohi s t oc he m i s t r y ( I H C ) ......................................11
S t a t i s t i c a l a na l ys e s .......................................................................................................15
C ha pt e r 2: R e s ul t ...........................................................................................................................16
D H M a m e l i or a t e s a nxi e t y- l i ke be ha vi or i n s oc i a l i s ol a t i on m i c e ................................16
T w o w e e ks s oc i a l i s ol a t i on do not i nf l ue nc e t he N F - κ B l e ve l i n br a i n.......................18
T he i s ol a t i on gr oup s how s a n i nc r e a s i ng t r e nd of hydr oge n pe r oxi de c onc e nt r a t i on
w hi l e D H M c a n r e ve r s e i t ............................................................................................20
D H M r e ve r s e s t he r e duc t i on of B D N F l e ve l s of m i c e e xpos e d t o s oc i a l
i s ol a t i on.....................................................................................................................21
D H M de c r e a s e s t he de ns i t y of a c t i va t e d a s t r oc yt e s i n s oc i a l i s ol a t i on m i c e ...............22
D H M m odul a t e s t he m or phol ogi c a l c ha nge s i n a s t r oc yt e a f t e r s oc i a l
i s ol a t i on.......................................................................................................................25
C ha pt e r 3: D i s c us s i on. ..................................................................................................................27
R e f e r e nc e s ................................................................................................……..............................31
i v
L i s t o f F i g u r e s
F i gu r e 1. T he c he m i c a l s t r uc t ur e of
di hydr om yr i c e t i n ……………………………………………2
F i gu r e 2. T he s ke l e t on a na l ys i s pr oc e s s f or a s t r oc yt e s by us i ng F i j i s of t w a r e ………………….14
F i gu r e 3. D H M a m e l i or a t e s a nxi e t y- l i ke be ha vi or i n s oc i a l i s ol a t i on
m i c e ……………………..16
F i gu r e 4. T w o w e e ks s oc i a l i s ol a t i on do not i nf l ue nc e t he N F - κ B l e ve l …………………….… .18
F i gu r e 5. T he i s ol a t i on gr oup s how s a n i nc r e a s i ng t r e nd of hydr oge n pe r oxi de c onc e nt r a t i on
w hi l e D H M c a n r e ve r s e
i t ………………………………………………………………………………20
F i gu r e 6. D H M r e ve r s e s t he r e duc t i on of B D N F pr ot e i n c a us e d by a nxi e t y ……………………21
F i gu r e 7. R e pr e s e nt a t i ve i m a ge s t a ke n by B i o- T e k C yt a t i on 5 C e l l I m a gi ng M ul t i - M ode R e a de r
a t a m a gni f i c a t i on of 20………………………………………………………………………….23
F i gu r e 8. D H M m odul a t e s t he m or phol ogi c a l c ha nge s i n a s t r oc yt e s a f t e r s oc i a l i s ol a t i on …….25
F i gu r e 9. R e pr e s e nt a t i ve m i c r ogl i a i m a ge s t a ke n by B i o- T e k C yt a t i on 5 C e l l I m a gi ng
M ul t i - M ode R e a de r a t a m a gni f i c a t i on of 20……………………………………………………30
v
A b b r e v i a t i o n s
B D N F B r a i n- de r i ve d ne ur ot r ophi c f a c t or
B S A B ovi ne s e r um a l bum i n
B Z s B e nz odi a z e pi ne s
C N S C e nt r a l ne r vous s ys t e m
D H M D i hydr om yr i c e t i n
G F A P G l i a l f i br i l l a r y a c i di c pr ot e i n
G A B A G a m m a - a m i nobut yr i c a c i d
G A B A A R G A B A A r e c e pt or
I H C I m m unohi s t oc he m i s t r y
L D 50 L e t ha l dos e 50%
P B S P hos pha t e buf f e r s ol ut i on
P F A P a r a f or m a l de hyde
S N R I s S e r ot oni n a nd N or e pi ne phr i ne R e upt a ke I nhi bi t or s
S S R I s S e l e c t i ve S e r ot oni n R e upt a ke I nhi bi t or s
T B S T r i s - B uf f e r e d S a l i ne
T r kB T yr os i ne ki na s e B
vi
A b s t r a c t
A nxi e t y di s or de r s a r e a s e r i e s of c hr oni c m e nt a l di s e a s e s t ha t a f f e c t m a ny pe opl e a l l
ove r t he w or l d, w hi c h a r e m a i nl y c ha r a c t e r i z e d by s i gni f i c a nt a nd unc ont r ol l a bl e f e e l i ngs of
a nxi e t y a nd de pr e s s e d. D ur i ng t he C O V I D - 19, pe opl e ’ s l i ve s ha ve be e n gr e a t l y a f f e c t e d.
L ong- t e r m qua r a nt i ne a t hom e a nd l a c k of s oc i a l a c t i vi t i e s ha ve l e d t o ge t t i ng a nxi e t y di s or de r s
m or e e a s i l y. D i hydr om yr i c e t i n ( D H M ) , a ki nd of bi oa c t i ve f l a vonoi d, s how s t he e f f e c t s on
de c r e a s i ng a l c ohol us a ge , r e duc i ng a nxi e t y- r e l a t e d be ha vi or s a nd r e pa i r i ng c ogni t i on i m pa i r m e nt
i n our t e a m ' s pr e vi ous s t udi e s . I n c ur r e nt s t udy, us i ng a s oc i a l i s ol a t i on m i c e m ode l e s t a bl i s he d i n
our l a b, w e f ound t ha t i s ol a t e d a ni m a l s s how e d i nc r e a s e d a nxi e t y l e ve l s i n e l e va t e d pl us m a z e
a nd ope n f i e l d be ha vi or a s s a ys . W e a l s o f ound a de c r e a s e d br a i n- de r i ve d ne ur ot r ophi c f a c t or
( B D N F ) l e ve l a s w e l l a s a c t i va t e d a s t r oc yt e s , gl i a l c e l l s w hi c h s t a bi l i z e s yna pt i c c onne c t i on a nd
m a i nt a i n s yna pt i c f unc t i on. W hi l e D H M c a n r e s t or e t he B D N F c onc e nt r a t i on t o a nor m a l l e ve l
a nd a l s o a l l e vi a t e s t he a c t i va t i on of a s t r oc yt e by de c r e a s i ng t he num be r of a s t r oc yt e a nd r e duc i ng
t he br a nc h num be r . O ur s t udy s ugge s t e d t ha t s hor t t e r m of s oc i a l i s ol a t i on c oul d i nduc e s
a s t r oc yt e ove r a c t i va t i on a nd B D N F r e duc t i on, bot h c ont r i but e t o s yna pt i c f unc t i ons . D H M c a n
r e s t or e t he s yna ps e dys f unc t i on a nd r e duc e a nxi e t y l e ve l , s ugge s t i ng D H M c a n be c ons i de r e d a s
a nove l c a ndi da t e f or t r e a t i ng a nxi e t y di s or de r s .
1
I n t r o d u c t i o n
A n xi e t y d i s or d e r s
A nxi e t y di s or de r s , i nc l udi ng f our ki nds of di s e a s e s ( ge ne r a l i z e d a nxi e t y di s or de r ,
obs e s s i ve - c om pul s i ve di s or de r , pa ni c di s or de r , s oc i a l a nxi e t y di s or de r a s w e l l a s pos t - t r a um a t i c
s t r e s s di s or de r ) a r e a s e r i e s of m os t pr e va l e nt ps yc hi a t r i c di s or de r s i n t he U ni t e d S t a t e s a nd e ve n
t he w or l d. B a s e d on t he da t a f r om a l a r ge popul a t i on- ba s e d s ur ve y, a bout 33.7% of t he
popul a t i on a r e a f f e c t e d by a nxi e t y di s or de r s dur i ng t he i r l i f e t i m e .[ 1] F ur t he r m or e , t he s e
di s or de r s a f f e c t a bout 18.1% of t he popul a t i on or 44 m i l l i on a dul t s i n t he U ni t e d S t a t e s e ve r y
ye a r . T he m a i n s ym pt om s of a nxi e t y di s or de r s a r e ne r vous ne s s a nd no- r e a s on a nxi e t y,
r e s t l e s s ne s s , a nd s ym pt om s of a ut onom i c dys f unc t i on, s uc h a s ha nd t r e m or s , s w e a t i ng a nd
f r e que nt ur i na t i on. A t pr e s e nt , t he c a us e of a nxi e t y di s or de r s i s not c l e a r a nd m a y be r e l a t e d t o
ge ne t i c f a c t or s , pe r s ona l i t y c ha r a c t e r i s t i c s , l i f e s t yl e , phys i c a l di s e a s e s a nd ot he r m e nt a l i l l ne s s e s .
A nxi e t y di s or de r s a r e a l s o hi ghl y c om or bi d w i t h ot he r ps yc hi a t r i c di s e a s e s s uc h a s bi pol a r
di s or de r , de pr e s s i ve di s or de r a nd e ve n A l z he i m e r ’ s di s e a s e .[ 2] S oc i a l i s ol a t i on i s de f i ne d a s a
s t a t e of l i t t l e or e ve n no c ont a c t w i t h a n i ndi vi dua l or s oc i e t y w hi c h ha s be e n r e c ogni z e d a s a
m a j or r i s k f a c t or f or m a ny m e nt a l i l l ne s s e s . [ 3] D ur i ng t he pa nde m i c t i m e , pe opl e a r e f or c e d t o
s pe nd m or e t i m e s t a yi ng a t hom e a nd ha ve l e s s c ha nc e t o c om m uni c a t e w i t h ot he r s f a c e t o f a c e .
A l a c k of s oc i a l c onne c t i on not onl y a f f e c t s m e nt a l he a l t h, but a l s o a f f e c t s phys i c a l he a l t h a nd
e ve n r e duc e s l i f e e xpe c t a nc y.[ 4]
T he di a gnos t i c s t a nda r d f or a nxi e t y di s or de r s i n t he U S i s t he D i a gnos t i c a nd S t a t i s t i c a l
M a nua l of M e nt a l D i s or de r s – F i f t h E di t i on ( D S M - V ) . [ 5] A l t hough m a ny pe opl e a r e pl a gue d by
a nxi e t y di s or de r , t he c ur r e nt t r e a t m e nt s a r e not s a t i s f a c t or y e nough. B e nz odi a z e pi ne s ( B Z s ) s uc h
a s di a z e pa m , l or a z e pa m or a l pr a z ol a m a r e c ons i de r e d a s t he f i r s t l i ne t r e a t m e nt s f or a nxi e t y
2
di s or de r but m a ny s i de e f f e c t s s uc h a s dr ow s i ne s s , c ogni t i on i m pa i r m e nt , s hor t dur a t i on of
c ur a t i ve e f f e c t , a nd dr ug r e s i s t a nc e a f t e r l ong- t e r m us e l i m i t i t s c l i ni c a l a ppl i c a t i on, B e s i de s
be nz odi a z e pi ne s , ot he r ki nds of dr ugs s uc h a s S e l e c t i ve S e r ot oni n R e upt a ke I nhi bi t or s ( S S R I s )
a nd S e r ot oni n a nd N or e pi ne phr i ne R e upt a ke I nhi bi t or s ( S N R I s ) ha ve de ve l ope d a s a l t e r na t i ve
t he r a pi e s f or a nxi e t y di s or de r s . H ow e ve r , s i m i l a r t o B Z s , t he y a l s o i ne vi t a bl y ha ve s om e a dve r s e
e f f e c t s l i ke f e e l i ng a gi t a t e d, i nc r e a s e d s e l f - s ui c i de t hought s a nd poor pa t i e nt c om pl i a nc e .[ 6] [ 7]
T he r e f or e , i t i s ne c e s s a r y t o de ve l op ot he r e f f e c t i ve a nd s a f e t r e a t m e nt m e t hods f or a nxi e t y
di s or de r s .
D i h yd r om yr i c e t i n ( D H M ) an d G A B A r e c e p t or
D i hydr om yr i c e t i n [ ( 2R ,3R ) - 3,5,7- t r i hydr oxy- 2- ( 3,4,5- t r i hydr oxyphe nyl ) - 2,3-
di hydr oc hr om e n- 4] i s a f l a vonoi d c om pound of A m pe l ops i s gr os s e de nt at a w hi c h i s m a i nl y
di s t r i but e d i n E a s t A s i a n a r e a a nd i s us e d a s he r ba l t e a f or dr i nki ng t o t r e a t f e ve r a nd
c ough.[ 8] [ 9]
F i gu r e 1. T he c he m i c a l s t r uc t ur e of di hydr om yr i c e t i n.
T he pha r m a c oki ne t i c pa r a m e t e r s of D H M w e r e de t e r m i ne d by a r a t or a l a bs or pt i on ( 20 m g/ kg)
e xpe r i m e nt . I t s how e d t ha t t he m a x c onc e nt r a t i on of D H M i n bl ood i s 21.63 ± 3.62 ng/ m l a t
3
a ppr oxi m a t e l y 2.67 h a f t e r t he or a l a dm i ni s t r a t i on. T he l ow bi oa va i l a bi l i t y ( 4.02% ) of D H M c a n
be e xpl a i ne d by t he a bs or pt i on of D H M m a i nl y t hr ough a pa s s i ve di f f us i on m e c ha ni s m . [ 10]
T he r e i s not t oo m uc h r e s e a r c h on t he t oxi c i t y of D H M , but i n t he s e l i m i t e d s t udi e s , D H M ha s
be e n pr ove d a s no s e r i ous a dve r s e r e a c t i ons a nd t he or a l a dm i ni s t r a t i on L D 50 ( l e t ha l dos e 50% )
i n m i c e i s l a r ge r t ha n 5g/ kg. [ 11]
G a m m a - a m i nobut yr i c a c i d ( G A B A ) i s a n i m por t a nt i nhi bi t or y ne ur ot r a ns m i t t e r e xi s t i ng i n
our br a i n pl a yi ng a ke y r ol e i n t he r e gul a t i on of ne ur ona l t r a ns m i s s i on. R e duc e d G A B A a c t i vi t y
a nd dys f unc t i on of G A B A e r gi c t r a ns m i s s i on ha s be e n f ound i n a nxi e t y di s or de r s . [ 12]
B e nz odi a z e pi ne s a r e dr ugs a ppr ove d by t he F D A f or t he t r e a t m e nt of a nxi e t y di s or de r s a nd i t s
m e c ha ni s m i s e nha nc i ng t he bi ndi ng of G A B A a nd i t s r e c e pt or , r e s ul t i ng i n s e da t i ve a nd
a nxi ol yt i c pr ope r t i e s . W e ha ve i de nt i f i e d t ha t di hydr om yr i c e t i n a s a pos i t i ve a l l os t e r i c m odul a t or
of G A B A A r e c e pt or ( G A B A A R ) ha s be e n r e c ogni z e d a s a n e f f e c t i ve t r e a t m e nt m e t hod i n
r e duc i ng a l c ohol c ons um pt i on, i m pr ovi ng a c ut e a l c ohol i nt oxi c a t i on a nd a l l e vi a t i ng a l c ohol
w i t hdr a w a l s ym pt om s i nc l udi ng w i t hdr a w a l - i nduc e d a nxi e t y be ha vi or i n our t e a m ' s pr e vi ous
s t udi e s .[ 13] F ur t he r m or e , a c c or di ng t o our r e s e a r c h on t he c e nt r a l ne r vous s ys t e m ( C N S ) , D H M
ha s s how n a c e r t a i n a nxi ol yt i c e f f e c t on A l z he i m e r ’ s di s e a s e m ous e m ode l s ( T G 2576 a nd
T G - S w D I ) by r e s t or i ng ge phyr i n l e ve l , dys f unc t i on of G A B A e r gi c ne ur ot r a ns m i s s i on a nd
f unc t i ona l s yna ps e s . T he unde r l yi ng m ol e c ul a r m e c ha ni s m i s a s s oc i a t e d w i t h i t s a bi l i t y t o
pr e ve nt t he c ha nge s i n s yna pt i c a nd e xt r a s yna pt i c G A B A A r e c e pt or s .[ 14] I n t he c l i ni c , a l t e r e d
G A B A e r gi c ne ur ot r a ns m i s s i on ha s a c l os e r e l a t i ons hi p w i t h t he pa t hophys i ol ogy of a nxi e t y
di s or de r s . T he r e f or e , w e ha ve t he r e a s on t o be l i e ve t ha t D H M ha s t he pos s i bi l i t y t o t r e a t a nxi e t y
di s or de r s .
4
B D N F
B r a i n- de r i ve d ne ur ot r ophi c f a c t or ( B D N F ) w hi c h i s w i de l y di s t r i but e d t hr oughout t he
br a i n w i t h t he hi ghe s t e xpr e s s i on i n t he hi ppoc a m pus i s a m e m be r of t he ne ur ot r ophi n f a m i l y.
B D N F w or ks by he l pi ng t o ke e p t he s t r uc t ur e a nd f unc t i on of e xi s t i ng s yna ps e s a nd r e gul a t i ng
gr ow t h of ne w ne ur ons a nd s yna ps e s .[ 15] I t a c t s by bi ndi ng t o t yr os i ne ki na s e B ( T r kB ) on t he
pos t s yna pt i c m e m br a ne , w hi c h c a n e nha nc e t he phos phor yl a t i on l e ve l of T r kB a nd a c t i va t e s t he
R a s - M A P K pa t hw a y. F i na l l y, C A M P r e s pons e e l e m e nt bi ndi ng pr ot e i n ( C R E B ) pr om ot e s
ne ur ona l c e l l s ur vi va l a nd i nc r e a s e s s yna pt i c pl a s t i c i t y a nd ne ur oge ne s i s by i nc r e a s i ng t he
e xpr e s s i on of t he B D N F ge ne a nd t he a nt i - a popt ot i c pr ot e i n ge ne B C L - 2. [ 16] S t r e s s f ul l i f e
e ve nt s l i ke c hr oni c s oc i a l i s ol a t i on ha ve a s ubs t a nt i a l c a us a l a s s oc i a t i on w i t h de pr e s s i on a nd
a nxi e t y a nd m a ny s t udi e s ha ve pr ove n t ha t s t r e s s ha s t he a bi l i t y t o de c r e a s e t he e xpr e s s i on of
B D N F m R N A i n t he hi ppoc a m pus . F ur t he r m or e , ne ur ot r ophi n hypot he s i s i s m e nt i one d i n
s e ve r a l s t udi e s w hi c h pos t ul a t e s t ha t t he dow nr e gul a t i on of B D N F e xpr e s s i on i n t he br a i n m a y
l e a d t o s yna pt i c t r a ns m i s s i on i m pa i r m e nt a nd de c r e a s e i n ne ur oge ne s i s i n a nxi e t y s ubj e c t s .
[ 17] [ 18] H e nc e de pl e t i on of t he ne ur ot r ophi c f a c t or s e s pe c i a l l y B D N F ha s be e n l i nke d t o t he
pa t hol ogy of m a ny br a i n di s e a s e s a nd de ve l opi ng dr ugs f or t hi s t a r ge t a r e c ons i de r e d a s pot e nt i a l
t he r a pi e s f or ps yc hi a t r i c di s or de r s i nc l udi ng a nxi e t y. [ 19]
A s t r oc yt e
A s t r oc yt e s , ol i gode ndr oc yt e s a nd m i c r ogl i a a r e t hr e e ki nds of gl i a l c e l l s i n t he c e nt r a l
ne r vous s ys t e m . A m ong a l l t hr e e gl i a l c e l l s , a s t r oc yt e s a r e t he m os t w i de l y di s t r i but e d a nd ha ve
i m por t a nt m e t a bol i c a nd s uppor t f unc t i ons i n t he br a i n, i nc l udi ng r e gul a t i ng t he i oni c
e nvi r onm e nt , pa r t i c i pa t i ng i n t he f or m a t i on of t he bl ood- br a i n ba r r i e r , a nd r e gul a t i ng s yna pt i c
5
a c t i vi t y.[ 20] I n t he pa s t , a s t r oc yt e s ha ve t r a di t i ona l l y be e n know n t o s uppor t ne ur ons , but t he y
a r e r e c e nt l y r e c ogni z e d a s r e gul a t i ng s yna ps e ne ur ot r a ns m i s s i on a nd pl a s t i c i t y.[ 21] M ul t i pl e
c e nt r a l ne r vous s ys t e m di s or de r s ha ve a c l os e r e l a t i on w i t h s yna pt i c i m pa i r m e nt w hi c h m a ni f e s t s
a s t he de c r e a s e d num be r of s yna ps e s a nd t he c ha nge of t he i r m or phol ogy. I n or de r t o m a i nt a i n
t he nor m a l f unc t i on a nd s t r uc t ur e of s yna ps e s , a s t r oc yt e s t e nd t o s how a n i nc r e a s i ng num be r a nd
l onge r br a nc he s , w hi c h i s c a l l e d a s t r oc yt e a c t i va t i on. T hi s phe nom e non i s a l s o know n a s r e a c t i ve
gl i os i s m a ni f e s t e d by s t r ong c e l l pr ol i f e r a t i on, m or phol ogi c a l a nd f unc t i ona l a l t e r a nd e nha nc e d
e xpr e s s i on of G l i a l F i br i l l a r y A c i di c P r ot e i n ( G F A P ) w hi c h i s a pr i nc i pa l c om pone nt of a s t r oc yt e
t o m a i nt a i n m e c ha ni c a l s t r e ngt h a nd s ha pe .[ 22] I n ge ne r a l , t he a bnor m a l i t y of a s t r oc yt e s m a y
c ont r i but e t o t he pa t hoge ne s i s of a nxi e t y di s or de r s .[ 23]
C h a p t e r 1 . M a t e r i a l s a n d m e t h o d s
1.1 A n i m al s u b j e c t s an d s oc i al i s ol at i on m od e l
S i x- w e e k- ol d m a l e C 57B L / 6 m i c e ( C ha r l e s R i ve r L a bor a t or i e s , H ol l i s t e r , C A ) w e r e
hous e d i n t he vi va r i um w i t h a t w e l ve hour s da yl i ght / da r k c yc l e ( da yl i ght t i m e 12: 00pm t o
12: 00a m ) w i t h f r e e a c c e s s t o w a t e r a nd f ood. A l l t he a ni m a l e xpe r i m e nt s w e r e pe r f or m e d
a c c or di ng t o t he pr ot oc ol s a ppr ove d by t he U ni ve r s i t y of C a l i f or ni a L os A nge l e s ( U C L A )
I ns t i t ut i ona l A ni m a l C a r e a nd U s e C om m i t t e e . B e f or e t he be gi nni ng of e xpe r i m e nt , a l l
e xpe r i m e nt m i c e w e r e ha bi t ua t e d t o t he vi va r i um f or t w o da ys . T i s s ue bi oc he m i c a l a na l ys e s w e r e
c onduc t e d a t t he U ni ve r s i t y of S out he r n C a l i f or ni a ( U S C ) . S oc i a l i s ol a t i on i s a m e t hod t o e l i c i t
a nxi ous be ha vi or s i n a ni m a l m ode l s a nd s ym pt om s of a nxi e t y i n m i c e t e nd t o s how up i n 4 t o 6
w e e ks .[ 24] F or gr oup hous e m i c e , w e r e a r e d t w o or t hr e e m i c e i n e a c h t r a ns pa r e nt c a ge w i t h
e nough be ddi ng. A l l t he c a ge s w e r e pl a c e d on t he s he l f w i t hout a ny obs t a c l e be t w e e n t w o c a ge s .
6
F or t he s oc i a l i s ol a t i on m i c e , t he y w e r e r e a r e d s e pa r a t e l y i n e a c h c a ge a nd a l l t he c a ge s w e r e
w r a ppe d w i t h bl a c k pl a s t i c ga r ba ge ba gs t o e s t a bl i s h a s oc i a l i s ol a t i on- i nduc e d a nxi e t y m ode l .
A l l t he m i c e w e r e e i t he r gr oup hous e d or i s ol a t e d f or 2 w e e ks be f or e t he y got 2- w e e k t r e a t m e nt .
D ur i ng t he t r e a t m e nt , a l l t he m i c e r e m a i ne d i n t he i r or i gi na l f e e di ng c ondi t i on. A l l t he a ni m a l s
w e r e r a ndom l y di vi de d i nt o f our gr oups . T he de t a i l e d gr oupi ng s i t ua t i on i s a s f ol l ow s .
( 1) G + V : G r oup- hous e d m i c e w e r e r e a r e d f or 2 w e e ks w i t h a dm i ni s t r a t i on of s uc r os e a ga r a s
a ve hi c l e f or a not he r 2 w e e ks .
( 2) G + D : G r oup- hous e d m i c e w e r e r e a r e d f or 2 w e e ks w i t h a dm i ni s t r a t i on of 2m g/ kg D H M
a ga r f or a not he r 2 w e e ks .
( 3) I + V : I s ol a t e d m i c e w e r e r e a r e d f or 2 w e e ks w i t h a dm i ni s t r a t i on of s uc r os e a ga r a s a
ve hi c l e f or a not he r 2 w e e ks .
( 4) I + D : I s ol a t e d m i c e w e r e r e a r e d f or 2 w e e ks w i t h a dm i ni s t r a t i on of 2m g/ kg D H M a ga r f or
a not he r 2 w e e ks .
1.2 D r u g p r e p ar at i on an d ad m i n i s t r at i on
D i hydr om yr i c e t i n ( D H M ) ( H P L C pur i f i e d > 98% , M a s t e r H e r bs I nc ., P om ona , C A ) w a s
a dm i ni s t e r e d or a l l y onc e a da y ( 2 m g/ kg or 10 m g/ kg) f or 2 w e e ks . T o pr e pa r e t he ve hi c l e a ga r ,
4% a ga r ( I nvi t r oge n, U S A ) w a s a dde d i nt o 130 m l D I w a t e r a nd he a t e d by m i c r ow a ve unt i l a l l
t he a ga r w a s c om pl e t e l y di s s ol ve d. T he n 0.8% s uc r os e w a s a dde d. T o pr e pa r e t he dr ug a ga r ,
0 . 0 0 4 % D H M p o w d e r f o r t h e 2 m g / k g g r o u p w a s a d d e d a n d m i x e d w i t h s u c r o s e a t t h e s a m e t i m e .
A f t e r a l l t he s uc r os e a nd D H M w e r e di s s ol ve d, t he s ol ut i on w a s pour e d i nt o a pl a t e a nd w a i t e d
f or c ool i ng t o s ol i di f y. A ga r w a s f i na l l y c ut i nt o 0.5*0.5*0.5 c m a nd ke pt i n t he r e f r i ge r a t or f or
7
da i l y us e . T he dos e w a s c a l c ul a t e d ba s e d on t he a ve r a ge body w e i ght of t he m ous e , w hi c h e qua l s
25 gr a m s .
E ve r y a f t e r noon ( 1 P M ) , a l l t he f ood a nd w a t e r w e r e r e m ove d f r om e a c h c a ge . A n a ga r
c ube w a s pl a c e d on a s m a l l pl a t e a nd t he n put t he pl a t e i n t he c or ne r of e a c h c a ge . T o m a ke s ur e
e a c h gr oupe d m ous e r e c e i ve d one a ga r , t he y w e r e i s ol a t e d i n di f f e r e nt c a ge s a nd f e d. A f t e r
t r e a t m e nt , t he gr oupe d m i c e w e r e r e t ur ne d t o t he i r or i gi na l c a ge s t o ke e p gr oup hous i ng. A l l t he
m i c e w e r e obs e r ve d f or a bout 60 m i nut e s t o m a ke s ur e a l l t he m i c e a t e t he i r ow n a ga r c ube .
1.3 B e h avi or al t e s t
A nxi e t y- l i ke be ha vi or a l t e s t s w e r e s t a r t e d on t he ne xt da y a f t e r t he l a s t da y of t r e a t m e nt .
B e c a us e m i c e a r e noc t ur na l a ni m a l s , a l l t he be ha vi or a l t e s t s w e r e c onduc t e d a t 1 pm i n t he da r k
pha s e t o m a ke s ur e w e c a n ge t m or e a c c ur a t e da t a . B e ha vi or a l t e s t s w e r e pe r f or m e d w i t h i ndi r e c t
r e d l i ght t o r e duc e t he i nt e r f e r e nc e c a us e d by e xt e r na l f a c t or s . A l l t he m i c e w e r e r a ndom l y
s e pa r a t e d i nt o t hr e e gr oups . C hoos e t w o di f f e r e nt gr oups of m i c e f or t he be ha vi or a l t e s t e ve r y
da y, one gr oup f or ope n f i e l d a nd t he ot he r f or e l e va t e d pl us m a z e . T he e nt i r e t e s t l a s t e d f or t hr e e
da ys . T he vi de o w a s r e c or de d by a n I phone or D J I O s m o P oc ke t . W he n c ha ngi ng m i c e , s pr a y
a l c ohol a nd us e t i s s ue t o w i pe t he c a ge t o e l i m i na t e t he s m e l l of m i c e . A l l t he da t a w a s
c onduc t e d i n bl i nd m e t hod a nd e a c h vi de o w a s obs e r ve d by t w o di f f e r e nt pe opl e .
O p e n f i e l d T e s t
O pe n f i e l d i s one of be ha vi or t e s t s t o de t e r m i ne ge ne r a l a nxi e t y l e ve l s i n r ode nt s . T he
s i z e of t he ope n f i e l d box m a de f r om w hi t e opa que pl a s t i c i s 50c m ( l e ngt h) *50c m ( w i dt h) *38c m
( he i ght ) T he c e nt e r z one i s f i ne d a s a 20c m *20c m s qua r e i n t he c e nt e r a nd t he c or ne r z one r e f e r s
8
t o f our 10c m *10c m s qua r e a r e a s l oc a t e d i n f our c or ne r s . T he a ni m a l s w e r e pl a c e d i n t he c e nt e r
of t he box a nd a l l ow e d t o f r e e l y m ove f or t e n m i nut e s w hi l e be i ng r e c or de d by a c a m e r a . W e
c a l c ul a t e d t he t i m e m i c e s pe nt i n t he c e nt e r a nd f our c or ne r s s e pa r a t e l y, r e a r i ng t i m e s a nd a l s o
a na l yz e d t he t ot a l di s t a nc e t he y pa s s e d.
E l e vat e d P l u s M az e
T he e l e va t e d pl us m a z e t a s k w a s c a r r i e d out i n t he pl us - m a z e a ppa r a t us w hi c h w a s m a de
of bl a c k opa que pl a s t i c . T he a ppa r a t us c ons i s t s of t w o ope n a r m s ( 25 c m l e ngt h x 5c m w i dt h)
a nd t he t w o c l os e d a r m s ha ve t he s a m e s i z e . T he c e nt r a l pl a t f or m i s a s qua r e a r e a of 5 c m by 5
c m . T he r e i s a w a l l a bout 20 c m hi gh a r ound t he c l os e d a r m t o f or m a c l os e d s pa c e . D ur i ng t he
e xpe r i m e nt , t he e nt i r e m a z e w a s pl a c e d on a t r i pod a bout 50 c m a bove t he f l oor . F or e a c h t e s t ,
one m ous e w a s pl a c e d i n t he c e nt e r of t he m a z e a nd a l l ow e d t o e xpl or e t he m a z e f r e e l y f or 5
m i nut e s . T he l e ve l of a nxi e t y w a s de t e r m i ne d by e va l ua t i ng t he num be r of e nt r i e s i nt o t he ope n
a r m a nd t he dur a t i on t i m e i n ope n a nd c l os e a r m s .
1.4 We s t e r n b l ot s
P B S : 100 m l 10x P B S ( B i o- r a d, U S A ) i s di l ut e d w i t h 900 m l D I w a t e r .
T B S : 100 m l 10x T B S ( B i o- r a d, U S A ) i s di l ut e d w i t h 900 m l D I w a t e r .
P B S - T : A dd 1 m l T w e e n 20 ( S i gm a , U S A ) i n 1000 m l P B S buf f e r .
T B S - T : A dd 1 m l T w e e n 20 ( S i gm a , U S A ) i n 1000 m l T B S buf f e r .
B l oc ki ng B uf f e r : 0.6 g non- f a t m i l k pow de r ( B l ot t i ng - G r a de B l oc ke r , B i o- R a d) i s di s s ol ve d i n
10m l P B S - T s ol ut i on.
9
A bout 20 m g br a i n s a m pl e f r om hi ppoc a m pus a r e a t a ke n f r om e a c h m i c e w a s
hom oge ni z e d w i t h l ys i s buf f e r ( C e l l L yt i c M T C e l l L ys i s R e a ge nt , S i gm a , U S A ) ( 10 μ l f or 1 m g
br a i n t i s s ue s a m pl e ) a nd 1% pr ot e a s e i nhi bi t or ( P r ot e a s e I nhi bi t or C oc kt a i l , C a l bi oc he m , U S A )
by us i ng a n ul t r a s oni c t i s s ue hom oge ni z e r ( B r a ns on S oni c a t or , U S A ) . C ol l e c t e d t he s upe r na t a nt
a f t e r t he hom oge na t e w a s c e nt r i f uge d a t 13000 r pm f or 15 m i ns . P r ot e i n c onc e nt r a t i on w a s
c a l c ul a t e d by t he B C A P r ot e i n A s s a y ki t ( P i e r c e B C A P r ot e i n A s s a y K i t , T he r m of i s he r , U S A )
a c c or di ng t o t he m a nuf a c t ur e r ’ s i ns t r uc t i ons .
T he a dde d s a m pl e a nd di l ut i on r a t i o w e r e a dj us t e d a c c or di ng t o t he r e s ul t s of B C A . A l l
t he s a m pl e s w e r e a dde d i nt o E P t ube s a nd t he t ube s w e r e he a t e d i n a m e t a l ba t h a t 80 de gr e e f or
10 m i ns . T he n c e nt r i f uge d t he s a m pl e s ol ut i on a t 13000 r pm f or 15 m i ns . A bout 30 μ g of pr ot e i n
w a s a dde d i n e a c h w e l l of 10% s odi um dode c yl s ul pha t e - pol ya c r yl a m i de ge l e l e c t r ophor e s i s
( S D S - P A G E ) ( 10% M i ni - P R O T E A N T G X G e l s , B i o- R a d, U S A ) . W e s e t t he vol t a ge a t 80 V a t
f i r s t t o m a ke s ur e a l l t he s a m pl e s a r e c om pr e s s e d i nt o a l i ne a nd c ha nge d t o 120 V f or a not he r 60
m i ns r un. T r a ns f e r r e d t he ge l t o P V D F m e m br a ne ( T r a ns - B l ot T ur bo T r a ns f e r P a c k, B i o- R a d,
U S A ) by us i ng T r a ns - B l ot T ur bo T r a ns f e r S ys t e m ( B i o- R a d,U S A ) . T he t r a ns f e r r e d m e m br a ne
w a s bl oc ke d w i t h a 10 m l bl oc ki ng buf f e r f or one a nd a ha l f hour s . W a s he d t he m e m br a ne t hr e e
t i m e s w i t h 1xT B S - T f or 5 m i nut e s a nd t he n I nc uba t e w i t h pr i m a r y a nt i body ove r ni ght ( N F - κ B
p65 ( D 14E 12) X P ® R a bbi t m A b #8242 ( 1: 1000) , B D N F A nt i body #47808( 1: 1000) , β - A c t i n
( 8H 10D 10) M ous e m A b #3700 ( 1: 1000) , C e l l s i gna l i ng T e c hnol ogy, U S A ) .
O n t he ne xt da y, t he m e m br a ne w a s w a s he d t hr e e t i m e s w i t h 1xT B S - T f or 5 m i nut e s a nd
i nc uba t e d w i t h a nt i - m ous e or a nt i - r a bbi t s e c onda r y a nt i body ( G oa t A nt i - R a bbi t I gG ( H + L ) - H R P
C onj uga t e ( 1: 3000) , G oa t A nt i - M ous e I gG ( H + L ) - H R P C onj uga t e ( 1: 3000) , B i o- R a d, U S A ) f or 1
hour . T he i m a ge s w e r e t a ke n w i t h e nha nc e d c he m i l um i ne s c e nc e de t e c t i on r e a ge nt ( C l a r i t y
1 0
W e s t e r n E C L S ubs t r a t e , B i o- R a d, U S A ) a nd C he m i - D oc ( B i o- R a d, U S A ) i m a gi ng de vi c e . A f t e r
i m a gi ng, us i ng a s t r i ppi ng buf f e r ( R e s t or e ™ W e s t e r n B l ot S t r i ppi ng B uf f e r , T he r m o s c i e nt i f i c ,
U S A ) t o r e m ove pr i m a r y a nd s e c onda r y a nt i bodi e s a nd i nc uba t e a not he r pr i m a r y a nt i body.
1.5 B D N F E L I S A
T he br a i n s a m pl e s w e r e hom oge ni z e d w i t h t he R I P A buf f e r ( T he r m of i s he r , U S A ) ( 10 μ l
f or 1m g br a i n t i s s ue s a m pl e ) . T he s upe r na t a nt of t he br a i n hom oge na t e w a s c ol l e c t e d a f t e r
c e nt r i f uga t i on a t 12000 r pm f or 10 m i nut e s . T he c onc e nt r a t i on of B D N F w a s de t e r m i ne d by t he
T ot a l B D N F Q ua nt i ki ne E L I S A K i t ( C a t a l og N um be r : D B N T 00, R & D s ys t e m s , U S A ) . A l l t he
s a m pl e s w e r e ne c e s s a r y t o be di l ut e d f i ve t i m e s w i t h t he C a l i br a t or D i l ue nt R D 5K buf f e r
pr ovi de d by t he ki t a nd a l l t he ot he r pr oc e s s e s f ol l ow e d t he ki t pr ot oc ol . T he r e s ul t w a s r e a d by
S yne r gy H 1 H ybr i d M ul t i - M ode M i c r opl a t e R e a de r ( B i o- T e k, U S A ) a t 450 nm w i t h 540 nm
w a ve l e ngt h c or r e c t i on.
1.6 H yd r oge n p e r oxi d e as s ay
T he c onc e nt r a t i on of hydr oge n pe r oxi de w a s de t e r m i ne d by us i ng a H ydr oge n P e r oxi de
A s s a y K i t ( A bc a m a b102500, M A , U S A ) . A l l t he e xpe r i m e nt pr oc e s s f ol l ow s t he ki t pr ot oc ol . I n
br i e f , a bout 20 m g br a i n s a m pl e s w e r e hom oge ni z e d m a nua l l y w i t h a 200 μ l hydr oge n pe r oxi de
a s s a y buf f e r pr ovi de d i n t hi s ki t . T he s upe r na t a nt w a s c ol l e c t e d a f t e r c e nt r i f uge d a t 13000 x g f or
5 m i ns . T he s a m pl e s w e r e de pr ot e i ni z e d us i ng 4M pe r c hl or i c a c i d f ol l ow e d by c e nt r i f uga t i on a t
13000 x g f or 2 m i ns . E xc e s s pe r c hl or i c a c i d w a s ne ut r a l i z e d by 2M pot a s s i um hydr oxi de unt i l
t he pH e qua l e d 6.5 t o 8. A l l t he s a m pl e s upe r na t a nt s w e r e c ol l e c t e d a f t e r c e nt r i f uga t i on. I n a
1 1
f l uor om e t r i c 96- w e l l pl a t e , 50 μ l of r e a c t i on m i x a s s a y buf f e r a nd 50 μ l of s a m pl e or s t a nda r d
w e r e a dde d i nt o e a c h w e l l . T he f l uor e s c e nc e i nt e ns i t y w a s r e a d by S yne r gy H 1 H ybr i d
M ul t i - M ode M i c r opl a t e R e a de r ( B i o- T e k, U S A ) s e t t i ng t he e xc i t a t i on w a ve l e ngt h a t 535 nm a nd
t he e m i s s i on w a ve l e ngt h a t 587 nm .
1.7 B r ai n T i s s u e P r e p ar at i on an d I m m u n oh i s t oc h e m i s t r y ( I H C )
W he n ne c r ops y, t he l e f t br a i n of m i c e w a s r e m ove d ge nt l y f r om t he s kul l . T he br a i ns w e r e
pos t f i xe d i n 10% pa r a f or m a l de hyde ( P F A ) ( S i gm a - A l dr i c h, U S A ) f or 24 hour s a t r oom
t e m pe r a t ur e a nd t he n t r a ns f e r r e d t o 30% s uc r os e ( S i gm a - A l dr i c h, U S A ) phos pha t e buf f e r s a l i ne
( P B S ) s ol ut i on ( R oc he , U S A ) f or c r yopr ot e c t i on. A f t e r 48 hour s , t he br a i ns w e r e w a s he d t hr e e
t i m e s by c ol d P B S a nd w e r e f r oz e n i m m e di a t e l y i n i s ope nt a ne , w hi c h w a s c ool e d by l i qui d
ni t r oge n. T he br a i ns w e r e s t or e d i n a - 80 de gr e e r e f r i ge r a t or f or f ur t he r us e .
F r oz e n t i s s u e as t r oc yt e s t ai n i n g
10% B S A : 1.2g bovi ne s e r um a l bum i n ( B S A ) ( S i gm a , U S A ) i s di s s ol ve d i n 12m l P B S - T .
B l oc ki ng buf f e r : T o pr e pa r e 5 m l bl oc ki ng buf f e r , 200μ l goa t s e r um a nd 1000ul 10% B S A a r e
a dde d i nt o 3800μ l P B S - T .
F or br a i n t i s s ue s l i c i ng, br a i n s e c t i ons c ont a i ni ng t he hi ppoc a m pus w e r e m ount e d i n
O .C .T ( S a kur a , U S A ) i n a s m a l l pl a t e on dr y i c e . 30 μ m - t hi c k s a gi t t a l br a i n s l i c e s w e r e s e c t i one d
by us i ng M i c r om H M 255 C r yos t a t m i c r ot om e a nd t r a ns f e r r e d on S upe r F r os t s l i de s ( V W R ,
U S A ) . A f t e r be i ng bl oc ke d w i t h t he bl oc ki ng buf f e r f or one da y, t he s l i de s w e r e i nc uba t e d w i t h
pr i m a r y a nt i body ( G F A P m ous e #3670 1: 500, C e l l s i gna l i ng, U S A ) i n a hum i di f i e r f or t w o da ys .
1 2
T he s l i de s w e r e w a s he d t hr e e t i m e s by P B S t o e l i m i na t e unbound a nt i bodi e s a nd i nc uba t e d
a n o t h e r d a y w i t h s e c o n d a r y a n t i b o d y ( g o a t a n t i - m o u s e V e c t o r D y L i g h t 5 5 0 1 : 2 5 0 , T h e r m o F i s h e r ,
U S A ) . [ 25] [ 26] F i na l l y, w e m ount e d t he s l i de s by us i ng t he D A P I m ount i ng m e di um ( A bc a m ,
U S A ) t o de t e r m i ne t he nuc l e us .
F r e e - F l oat i n g m i c r ogl i a s t ai n i n g
10% T r i t on- X 100 s ol ut i on: A dd 5 m l T r i t on- X 100 ( S i gm a - A l dr i c h, U S A ) i nt o 45 m l D I w a t e r .
T r i s - T r i t on: F or 1 L T r i s - T r i t on buf f e r , 100 m l 10x T B S ( B i o- r a d, U S A ) i s di l ut e d w i t h 900m l
D I w a t e r . T he n a dd 500 μ l 10% T r i t on- X 100 a nd a dj us t t he pH t o 7.4.
B l oc ki ng buf f e r : F or e a c h w e l l , pr e pa r e 1 m l bl oc ki ng buf f e r . A dd 20 ul 10% T r i t on- X 100 i nt o
1000 μ l T r i s - T r i t on a nd t he n a dd 20 μ l nor m a l goa t s e r um .
F or br a i n t i s s ue s l i c i ng, br a i n s e c t i ons c ont a i ni ng t he hi ppoc a m pus w e r e m ount e d i n O .C .T
( S a kur a , U S A ) on dr y i c e . 30 μ m - t hi c k s a gi t t a l br a i n s l i c e s w e r e s e c t i one d by us i ng M i c r om H M
255 C r yos t a t m i c r ot om e . A l l t he br a i n t i s s ue w a s t r a ns f e r r e d t o a 12- w e l l pl a t e f i l l e d w i t h c ol d
P B S a nd t he r e w e r e a bout 3- 4 pi e c e s of br a i n s a m pl e i n one w e l l . T he s a m pl e s w e r e r i ns e d t hr e e
t i m e s a nd t he n w a s he d one t i m e f or 10 t i m e s on a s ha ke r by T r i s - T r i t on. T i s s ue w a s i nc uba t e d i n
a bl oc ki ng buf f e r f or 1 hour a nd c ont i nuous l y i nc uba t e d w i t h pr i m a r y a nt i body ( A nt i - I ba - 1 r a bbi t
1: 500, F uj i f i l m W a ko, U S A ) f or t hr e e da ys a t 4 de gr e e . O n t he t hi r d da y, w e w a s he d t he s l i de s
t hr e e t i m e s w i t h T r i s - T r i t on a nd t he n i nc uba t e d t he m w i t h s e c onda r y a nt i bodi e s ( goa t a nt i - m ous e
V e c t or D yL i ght 550 1: 250, T he r m oF i s he r , U S A ) f or 30 m i nut e s . F i na l l y, a f t e r w a s hi ng t hr e e
t i m e s w i t h T r i s - T r i on, t he s l i de s w e r e m ount e d by t he D A P I m ount i ng m e di um ( A bc a m , U S A ) .
K e e p t he s l i de s i n r e f r i ge r a t i on f or f ur t he r i m a gi ng.
1 3
I m age ac q u i s i t i on an d an al ys i s
A l l t he i m a ge s of br a i n t i s s ue w e r e t a ke n by a f l uor e s c e nc e m i c r os c ope ( C yt a t i on 5 C e l l
I m a ge , B i oT e k, U S A ) w i t h z - s t a c k f unc t i on. F or t he C A a nd D G a r e a s of e a c h t i s s ue s e c t i on, 3
i m a ge s w e r e t a ke n s e pa r a t e l y a t 20x m a gni f i c a t i on. T he n i m a ge s of D A P I a nd a s t r oc yt e w e r e
m e r ge d by P hot os hop 2020 ( A dobe , U S A ) , w hi c h w a s a l s o us e d f or c a l c ul a t i ng t he c e l l de ns i t y.
F or c ount i ng t he c e l l de ns i t y, us e t he l a s s o t ool t o r a ndom l y s e l e c t a n a r e a a nd r e c or d t he t ot a l
num be r of pi xe l s i n t he s e l e c t e d a r e a . S ubs e que nt l y, c ount t he num be r of c e l l s w i t hi n t hi s a r e a .
F i ve a r e a s w e r e r a ndom l y s e l e c t e d f or e a c h i m a ge .
1 4
F i gu r e 2. T he s ke l e t on a na l ys i s pr oc e s s f or a s t r oc yt e s by us i ng F i j i s of t w a r e . A . A n or i gi na l i m a ge t a ke n by B i o- T e k C yt a t i on 5
C e l l I m a gi ng M ul t i - M ode R e a de r w i t h 20x m a gni f i c a t i on. B . C ha nge t he i m a ge t o 8- bi t a nd c onve r t t o gr a ys c a l e . C . A dj us t t he
br i ght ne s s a nd c ont r a s t . D . C onve r t t he i m a ge t o bi na r y us i ng t hr e s hol d f unc t i on. E . R e m ove s i ngl e - pi xe l ba c kgr ound noi s e a nd
out l i e r . F . S ke l e t oni z e t he i m a ge . S c a l e ba r e qua l s 100 μ m
M or phol ogy of a s t r oc yt e w a s a na l yz e d w i t h t he a i d of F i j i s of t w a r e ( N I H , B e t he s da ,
M D , U S A ) . S ke l e t on a na l ys i s i s a m e t hod t o a na l yz e t he m or phol ogy of gl i a l c e l l s . I n s hor t ,
1 5
f i r s t l y, t he i m a ge s t a ke n by B i o- T e k C yt a t i on 5 C e l l I m a gi ng M ul t i - M ode R e a de r w e r e c onve r t e d
i nt o 8- bi t gr a ys c a l e i m a ge s . B y a dj us t i ng t he br i ght ne s s a nd c ont r a s t , gl i a l c e l l s c a n be obs e r ve d
m or e c l e a r l y. A f t e r r e m ovi ng t he ba c kgr ound noi s e a nd t hr e s hol d, t he y w e r e f i na l l y t r a ns f or m e d
i nt o s ke l e t on pi c t ur e s . U s e d s ke l e t on a na l ys i s t o ge t t he r e qui r e d pa r a m e t e r s s uc h a s t he num be r
a nd l e ngt h of t he a ve r a ge br a nc h.[ 27]
1.8 S t at i s t i c al an al ys e s
D a t a a r e e xhi bi t e d a s t he m e a n ± S E M . A l l da t a a na l ys i s w a s pe r f or m e d w i t h P r i s m
s of t w a r e ve r s i on 6.0 ( G r a phP a d S of t w a r e I nc ., L a J ol l a , C A ) us i ng O ne - w a y A N O V A f ol l ow e d
by m ul t i pl e c om pa r i s on a na l ys e s . P va l ue l e s s t ha n 0.05 i s c ons i de r e d a s a s i gni f i c a nt di f f e r e nc e .
1 6
C h a p t e r 2 . R e s u l t s
D H M am e l i or at e s an xi e t y- l i k e b e h avi or i n s oc i al i s ol at i on m i c e
F i gu r e 3. D H M a m e l i or a t e s a nxi e t y- l i ke be ha vi or i n s oc i a l i s ol a t i on m i c e . ( A ) I n t he e l e va t e d pl us m a z e , t he e f f e c t s of s oc i a l
i s ol a t i on a nd D H M t r e a t m e nt w e r e m e a s ur e d by t hr e e pa r a m e t e r s ( t i m e s pe nt i n ope n a r m , c l os e a r m a nd t he a r e a be t w e e n t w o
c r os s a r m s c a l l e d i nt e r s e c t i on t i m e ) . O ne - W a y A N O V A f ol l ow e d by m ul t i pl e c om pa r i s ons , H ol m - S i da k m e t hod. F or ope n a r m s ,
p = 0.0439. F or c l os e a r m s , p = 0.0317. F or i nt e r s e c t i on, p = 0.9867 vs . ve hi c l e gr oup hous i ng c ont r ol ( G + V ) . ( B ) I n t he ope n
f i e l d e xpe r i m e nt , t he e f f e c t s of s oc i a l i s ol a t i on a nd t r e a t m e nt be ha vi or w e r e m e a s ur e d by r unni ng di s t a nc e ( t ot a l di s t a nc e of
1 7
m ovi ng) , r e a r i ng t i m e s ( t ot a l num be r of r e a r i ng t i m e s ) , c e nt e r t i m e ( t he t ot a l t i m e dur a t i on m i c e s pe nt i n t he c e nt e r ) , a nd c or ne r
( t he t ot a l dur a t i on m i c e s t a ye d i n t he f our c or ne r 10x10 c m s qua r e s ) . O ne - W a y A N O V A f ol l ow e d by m ul t i pl e c om pa r i s ons ,
H ol m - S i da k m e t hod. F or r unni ng di s t a nc e p = 0.0022. F or num be r s of r e a r i ngs , p = 0.0129. F or s t a y i n t he c e nt e r , p = 0.0313.
F or s t a y i n c or ne r s p < 0.0001. vs . ve hi c l e hous i ng c ont r ol gr oup ( G + V ) . ( n = 8/ gr oup) . G + V : gr oupe d m i c e w i t h ve hi c l e
t r e a t m e nt ; G + D : gr oupe d m i c e w i t h 2 m g/ kg D H M t r e a t m e nt ; I + V : s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ; I + D : s oc i a l
i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt . D a t a w e r e a l l s how n a s m e a n ± S E M . * p < 0.05, **p < 0.01, *** p < 0.001, ****p
< 0.0001
I n t he 5- m i nut e e l e va t e d pl us m a z e t e s t , gr oup- hous e d c ont r ol m i c e ( G + V ) s pe nt
2.476± 0.315 m i nut e s i n t he ope n a r m s a nd 2.241± 0.309 m i nut e s i n t he c l os e d a r m . M i c e s oc i a l l y
i s ol a t e d f or t w o w e e ks , f ol l ow e d by t w o w e e ks of ve hi c l e t r e a t m e nt ( I + V ) s pe nt l e s s t i m e
( 1.613 ± 0.263 m i n) i n t he ope n a r m s a nd m or e t i m e i n t he c l os e d a r m s ( 3.313 ± 0.265 m i n) . T he
t r e a t m e nt m i c e ( I + D ) s pe nt l onge r t i m e i n t he ope n a r m ( 2.269 ± 0.267 m i n) w he n c om pa r e d w i t h
unt r e a t e d s oc i a l l y i s ol a t e d m i c e . T he r e i s no di f f e r e nc e i n i nt e r s e c t i on t i m e . W e c a n obs e r ve t ha t
t he m i c e i n t he s oc i a l i s ol a t i on gr oup w e r e m or e i nc l i ne d t o s pe nd t i m e s t a yi ng i n t he c l os e d
a r m s , w hi c h i ndi c a t e s t ha t s oc i a l i s ol a t i on c a n i nc r e a s e a nxi e t y l e ve l . F ur t he r m or e , a f t e r
a dm i ni s t e r e d w i t h D H M , i s ol a t i on m i c e s pe nt m or e t i m e i n t he ope n a r m a nd s how e d t he s a m e
l e ve l a s t he c ont r ol gr oup ( p = 0.9517) , w hi c h pr ove d D H M c a n a m e l i or a t e i s ol a t i on- i nduc e d
a nxi e t y be ha vi or .
O pe n f i e l d w a s de s i gne d a s a not he r m e t hod t o f ur t he r e xa m i ne a nxi e t y- l i ke be ha vi or . I n
t he t ot a l t e n m i nut e s t e s t , m i c e i n t he c ont r ol gr oup ( G + V ) e xpl or e d f ur t he r di s t a nc e s ( 3385 ± 189
c m ) t ha n i s ol a t e d m i c e ( I + V ) w hi c h de m ons t r a t e d a s i gni f i c a nt r e duc t i on ( 2308 ± 145 c m ) i n
a ve r a ge l oc om ot i on di s t a nc e . H ow e ve r , t he s e a nxi e t y be ha vi or a l r e s pons e s c oul d be a m e l i or a t e d
by D H M a nd t he a ve r a ge di s t a nc e i n t he t r e a t m e nt gr oup ( I + D ) w a s i nc r e a s e d t o 2904± 286 c m .
R e a r i ng i s c ons i de r e d a s one of t he e xpl or a t or y be ha vi or s i n r ode nt s . T he num be r of r e a r i ng w a s
s i gni f i c a nt l y de c r e a s e d i n t he s i ngl e - hous e d m i c e c om pa r e d w i t h gr oup- hous e d m i c e ( 29.85 ± 1.97
t i m e s vs 49.08± 2.55 t i m e s ) . T he num be r of r e a r i ng i nc r e a s e d t o 42.63± 6.79 t i m e s i n t he D H M
1 8
t r e a t m e nt gr oup ( I + D ) . F or t he c or ne r a nd c e nt e r t i m e , c om pa r e d w i t h gr oup- hous e d m i c e ,
i s ol a t i on m i c e s pe nt i n t he c or ne r s w e r e s i gni f i c a nt l y i nc r e a s e d ( 71.13 ± 6.31 s e c vs 27.30± 2.31
s e c ) a nd t he t i m e i n t he c e nt e r de c r e a s e d f r om 18.43± 2.15 s e c t o 9.00± 1.83 s e c onds . A f t e r be i ng
t r e a t e d w i t h D H M , t he m i c e i n t he I + D gr oup s how e d s i m i l a r be ha vi or a l pa r a m e t e r s a s t he
c ont r ol gr oup, w hi c h m e a ns t he y w e r e w i l l i ng t o s pe nd m or e t i m e i n t he c e nt e r a nd l e s s t i m e i n
t he c or ne r . I n ge ne r a l , s oc i a l i s ol a t i on w a s pr ove d a s a n e f f e c t i ve m e t hod t o e l i c i t a nxi e t y i n m i c e
be c a us e i t c a n de c r e a s e t he e xpl or a t or y a c t i vi t y of m i c e a nd m a ke t he m s pe nd m or e t i m e s t a yi ng
i n t he c or ne r i ns t e a d of i n t he c e nt e r . H ow e ve r , a l l t he s e a nxi e t y- l i ke be ha vi or s c a n be r e ve r s e d
by t a ki ng D H M .
T w o w e e k s s oc i al i s ol at i on d o n ot i n f l u e n c e t h e N F - κ B l e ve l i n b r ai n
F i gu r e 4 . T w o w e e ks of s oc i a l i s ol a t i on do not i nf l ue nc e t he N F - κ B l e ve l ( A ) R e pr e s e nt a t i ve W e s t e r n bl ot i m a ge of N F - κ B a nd
B - a c t i n. T he hi ppoc a m pus s a m pl e s f r om e a c h gr oup of m i c e w e r e us e d. ( B ) T he ba r gr a ph i ndi c a t e s t he r e i s no s i gni f i c a nt
di f f e r e nc e be t w e e n e a c h gr oup ( n = 4) . B - a c t i n w a s us e d a s l oa di ng c ont r ol a nd G + V gr oup w a s c ons i de r e d a s c ont r ol gr oup.
1 9
G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; G + D : gr oupe d m i c e w i t h 2 m g/ kg D H M t r e a t m e nt ; I + V : s oc i a l i s ol a t i on m i c e w i t h
ve hi c l e t r e a t m e nt ; I + D : s oc i a l i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt . U s e O ne - W a y A N O V A t e s t . p = 0.1560. D a t a w e r e
a l l s how n a s m e a n ± S E M .
I nf l a m m a t i on i s a de f e ns i ve i m m une r e s pons e of t he body t o s t i m ul i . I n r e l a t i on t o t he
br a i n, ne ur oi nf l a m m a t i on i s a l w a ys di s c us s e d i n m a ny ne ur ode ge ne r a t i ve a nd ps yc hi a t r i c
di s or de r s s t udi e s l i ke A l z he i m e r di s e a s e , a ut i s m s pe c t r um di s or de r a nd de pr e s s i on. L ong- t e r m
s oc i a l i s ol a t i on c ons i de r e d a s a c hr oni c s t r e s s m a y a l s o c a us e ne ur oi nf l a m m a t i on i n a nxi e t y m i c e
m ode l . I n or de r t o c onf i r m t hi s hypot he s i s , w e c hos e N F - κ B a s a bi om a r ke r t o de t e c t be c a us e i t
i s a n i m por t a nt i ndi c a t or of i nf l a m m a t i on a nd i nvol ve d i n m a ny i nf l a m m a t i on c yt oki ne
e xpr e s s i ons .[ 28] [ 29] N F - κ B i s a nuc l e a r t r a ns c r i pt i on f a c t or e xpr e s s e d i n a l m os t a l l c e l l s a nd ha s
a c l os e r e l a t i ons hi p w i t h t he a c t i va t i on of m i c r ogl i a . I n t he c e nt r a l ne ur on s ys t e m , N F - κ B
a c t i va t i on c a n be s t i m ul a t e d by c a noni c a l , non- c a noni c a l a nd a t ypi c a l pa t hw a ys .[ 30] N F - κ B
pr ot e i n e xpr e s s i on i n m i c e hi ppoc a m pus w a s m e a s ur e d by W e s t e r n bl ot m e t hod. T he N F - κ B
de ns i t y w a s nor m a l i z e d by b- a c t i n w hi c h w or ks a s a l oa di ng c ont r ol pr ot e i n. T he f i r s t gr oup
( G + V ) w a s c ons i de r e d a s a c ont r ol gr oup a nd t he ot he r t hr e e gr oups w e r e c om pa r e d w i t h t he
c ont r ol gr oup t o ge t t he f i na l r e l a t i ve r a t i o. C om pa r e d w i t h t he c ont r ol gr oup, t he r e l a t i ve r a t i o
f or i s ol a t e d m i c e i s 0.7735± 0.093 w hi l e t he t r e a t m e nt gr oup i s 0.8033± 0.1453. T he r e i s no
s i gni f i c a nt di f f e r e nc e be t w e e n e a c h t w o gr oups p = 0.1560 by us i ng O ne - W a y A N O V A a na l ys i s .
T he r e f or e , w e c a n c onc l ude t ha t a nxi e t y c a us e d by s oc i a l i s ol a t i on m a y not be r e l a t e d t o N F - κ B
pa t hw a y .
2 0
T h e i s ol at i on gr ou p s h ow s an i n c r e as i n g t r e n d of h yd r oge n p e r oxi d e c on c e n t r at i on w h i l e
D H M c an r e ve r s e i t
F i gu r e 5 . T he i s ol a t i on gr oup s how s a n i nc r e a s i ng t r e nd of hydr oge n pe r oxi de c onc e nt r a t i on w hi l e D H M c a n r e ve r s e i t . T he
c onc e nt r a t i on of hydr oge n pe r oxi de i n m i c e br a i n s a m pl e s w a s de t e r m i ne d by us i ng a H ydr oge n P e r oxi de A s s a y K i t ( A bc a m
a b102500, M A , U S A ) . n = 3 G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; G + D : gr oupe d m i c e w i t h 2m g/ kg D H M t r e a t m e nt ; I + V :
s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ; I + D : s oc i a l i s ol a t i on m i c e w i t h 2m g/ kg D H M t r e a t m e nt . U s e O ne - W a y A N O V A
f ol l ow e d by m ul t i pl e c om pa r i s ons t o a na l ys i s . D a t a w e r e a l l s how n a s m e a n ± S E M . ( p = 0.1154 > 0.05)
S om e s t udi e s ha ve poi nt e d out t ha t m a ny ps yc hi a t r i c di s or de r s i nc l udi ng a nxi e t y ha ve a
c l os e r e l a t i ons hi p w i t h oxi da t i ve s t r e s s , e s pe c i a l l y r e a c t i ve oxyge n s pe c i e s ( R O S ) . H ydr oge n
pe r oxi de i s a r e a c t i ve oxyge n m e t a bol i c by- pr oduc t w hi c h s e r ve s a s a ke y r e gul a t or f or oxi da t i ve
s t r e s s - r e l a t e d s t a t e s . I t w i l l c a us e da m a ge t o t he br a i n w he n i t s l e ve l e xc e e ds t he a nt i oxi da t i ve
c a pa c i t y of c a t a l a s e a nd gl ut a t hi one pe r oxi da s e .[ 31] I n or de r t o m e a s ur e t he c onc e nt r a t i on of
hydr oge n pe r oxi de i n m i c e br a i n t i s s ue , w e us e d a H ydr oge n P e r oxi de A s s a y K i t ( A bc a m
a b102500, M A , U S A ) . B a s e d on t he r e s ul t of t he O ne - W a y A N O V A t e s t ( p = 0.1154) , t he r e i s
no s i gni f i c a nt di f f e r e nc e i n hydr oge n pe r oxi de be t w e e n a ny t w o gr oups . H ow e ve r , c om pa r e d
w i t h t he c ont r ol gr oup ( 0.082 ± 0.005 nm ol / m g ) , t he c onc e nt r a t i on of hydr oge n pe r oxi de i n t he
i s ol a t i on gr oup s how e d a n upw a r d t r e nd ( 0.1078 ± 0.0138 nm ol / m g ) w hi l e t hi s phe nom e non c a n
2 1
be r e ve r s e d by D H M w or ke d a s a n a nt i oxi da nt s . W e c a n obs e r ve t ha t i n t he t r e a t m e nt gr oup
( I + D ) t he c onc e nt r a t i on w a s r e duc e d t o 0.0835± 0.0044 nm ol / m g. A c onc l us i on c a n be a ddr e s s e d
t ha t s oc i a l i s ol a t i on c a n c a us e i nc r e a s e d l e ve l s of hydr oge n pe r oxi de i n t he a nxi e t y m i c e a nd t hi s
c a n be a l l e vi a t e d by D H M t r e a t m e nt but f ur t he r s t udi e s a r e ne e de d t o c onf i r m t hi s c onc l us i on.
D H M r e ve r s e s t h e r e d u c t i on of B D N F l e ve l s of m i c e e xp os e d t o s oc i al i s ol at i on
F i gu r e 6 . D H M r e ve r s e s t he r e duc t i on of B D N F pr ot e i n c a us e d by a nxi e t y. ( A ) R e pr e s e nt a t i ve i m a ge of B D N F W e s t e r n bl ot by
us i ng hi ppoc a m pus s a m pl e s t a ke n f r om e a c h t r e a t m e nt gr oup. B - a c t i n w a s us e d a s l oa di ng c ont r ol . ( B ) T he ba r gr a ph i ndi c a t e s
B D N F c onc e nt r a t i on i n m i c e br a i n s a m pl e s de t e r m i ne d by T ot a l B D N F Q ua nt i ki ne E L I S A K i t ( C a t a l og N um be r : D B N T 00,
R & D s ys t e m s , U S A ) . n = 3 G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; G + D : gr oupe d m i c e w i t h 2m g/ kg D H M t r e a t m e nt ; I + V :
s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ; I + D : s oc i a l i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt . U s e O ne - W a y A N O V A
f ol l ow e d by m ul t i pl e c om pa r i s ons t o a na l ys i s . D a t a w e r e a l l s how n a s m e a n ± S E M . * p < 0.05
B r a i n- de r i ve d ne ur ot r ophi c f a c t or ( B D N F ) i s a one of t he ne ur ot r ophi n f a m i l y m e m be r s
w hi c h i s hi ghl y di s t r i but e d i n t he c e nt r a l ne r vous s ys t e m . B D N F s uppor t s t he s ur vi va l a nd
m a i nt e na nc e of ne ur on c e l l s a nd ha s a c l os e a s s oc i a t i on w i t h ps yc hol ogi c a l di s e a s e . I n t he br a i n,
t he s ynt he s i s of B D N F i s m a i nl y l oc a t e d i n t he hi ppoc a m pus s o w e c hos e t o us e hi ppoc a m pus
2 2
s a m pl e s f or W e s t e r n B l ot s . B a s e d on t he r e s ul t , w e f ound t ha t t he B D N F l e ve l i n t he
hi ppoc a m pus w a s s i gni f i c a nt l y de c r e a s e d i n t he s oc i a l i s ol a t i on gr oup w hi l e t he D H M t r e a t m e nt
gr oup c a n r e ve r s e a l i t t l e bi t . I n or de r t o a c c ur a t e l y de t e r m i ne t he c onc e nt r a t i on of B D N F , w e
us e d t he B D N F Q ua nt i ki ne E L I S A K i t ( C a t a l og N um be r : D B N T 00, R & D s ys t e m s , U S A ) . I t
s how e d t he s a m e r e s ul t w i t h w e s t e r n bl ot . C om pa r e d w i t h t he c ont r ol gr oup, t he c onc e nt r a t i on of
B D N F i n i s ol a t i on w i t h t he ve hi c l e gr oup ( G + V ) r e duc e d f r om 5.711 pg/ m g t o 4.844 pg/ m g a nd
t he r e i s a s i gni f i c a nt di f f e r e nc e p = 0.0223. H ow e ve r , i n t he t r e a t m e nt gr oup ( I + D ) , t he B D N F
l e ve l w a s r e s t or e d t o 5.743 pg/ m g a nd a t t he s a m e t i m e D H M di d not i m pa c t t he B D N F
c onc e nt r a t i on i n gr oupe d m i c e . B a s e d on t he r e s ul t of W e s t e r n B l ot s a nd E L I S A , w e c a n i ndi c a t e
t ha t t he B D N F l e ve l de c r e a s e d s i gni f i c a nt l y i n s oc i a l i s ol a t i on m i c e c om pa r e d w i t h gr oupe d m i c e
w hi l e D H M w or ki ng a s a n a nxi ol yt i c dr ug c a n r e s t or e B D N F t o nor m a l l e ve l .
D H M d e c r e as e s t h e d e n s i t y of ac t i vat e d as t r oc yt e i n s oc i al i s ol at i on m i c e
2 3
F i gu r e 7. R e pr e s e nt a t i ve i m a ge s t a ke n by B i o- T e k C yt a t i on 5 C e l l I m a gi ng M ul t i - M ode R e a de r a t a m a gni f i c a t i on of 20. I m a ge s
s how G F A P s t a i ne d a s t r oc yt e s a nd D A P I s t a i ne d nuc l e us i n m i c e hi ppoc a m pus A . C or nu A m m oni s 1 ( C A 1) a nd B . D e nt a t e
G yr us ( D G ) r e gi on. S c a l e ba r e qua l s 100um . C . Q ua nt i f i c a t i on r e s ul t of a s t r oc yt e num be r pe r 10000 pi xe l s i n C A 1 a nd D G a e r a
s e pa r a t e l y. n = 3- 4 e a c h gr oup. G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; I + V : s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ;
I + D : s oc i a l i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt . U s e O ne - W a y A N O V A f ol l ow e d by m ul t i pl e c om pa r i s ons t o a na l ys i s .
D a t a w e r e a l l s how n a s m e a n ± S E M . * p < 0.05
2 4
G l i a l f i br i l l a r y a c i di c pr ot e i n ( G F A P ) i s t he s ke l e t a l pr ot e i n of a s t r oc yt e s t o m a i nt a i n t he i r
m e c ha ni c a l s t r e ngt h a nd s ha pe . U s i ng G F A P i s a n e f f e c t i ve m e t hod t o i m m unohi s t oc he m i c a l l y
di s t i ngui s h t he de ns i t y a nd m or phol ogy of a s t r oc yt e s f r om ot he r gl i a l c e l l s .[ 32] T he
hi ppoc a m pus i s one of t he m os t i m por t a nt s t r uc t ur e s i n t he br a i n w hi c h i s r e s pons i bl e f or bot h
s hor t - t e r m a nd l ong- t e r m m e m or y. I n m a ny pa t i e nt s w i t h m e nt a l i l l ne s s e s , e s pe c i a l l y A l z he i m e r ' s
di s e a s e , t he pa t hol ogi c a l c ha nge of t he hi ppoc a m pus i s one of t he m os t c l i ni c a l l y obs e r va bl e
phe nom e na be c a us e t he hi ppoc a m pus i s s us c e pt i bl e t o va r i ous e xoge nous a nd e ndoge nous
f a c t or s . T o obs e r ve t he t r e a t m e nt e f f e c t of D H M t o a c t i va t e d a s t r oc yt e s , w e s t a i ne d t he br a i n
t i s s ue w i t h G F A P a nt i body a nd m ount e d i t w i t h D A P I . T he i m a ge s of D G a nd C A 1 t a ke n by
m i c r os c ope w e r e m e r ge d i n P hot os hop a nd t he ove r l a ppe d a s t r oc yt e s w e r e c ount e d m a nua l l y.
A l l t he r e s ul t s w e r e s how n i n a s t r oc yt e num be r pe r 10000 pi xe l s . I n t he C A 1 r e gi on, w e c a n
i nve s t i ga t e t he i nc r e a s e d t r e nd of a s t r oc yt e de ns i t y i n 4- w e e k i s ol a t i on m i c e a nd D H M c a n
a l l e vi a t e t he r i s i ng l e ve l . T hi s phe nom e non w a s m or e obvi ous i n t he D G r e gi on. C om pa r e d w i t h
t he c ont r ol gr oup ( 0.5195 a s t r oc yt e pe r 10000 pi xe l s ) , t he num be r of a s t r oc yt e s i n t he i s ol a t i on
gr oup i nc r e a s e d t o 0.6611 pe r 10000 pi xe l s . H ow e ve r , a f t e r t w o w e e ks of D H M t r e a t m e nt , t he
num be r of a s t r oc yt e s i n t he I + D gr oup m i c e de c r e a s e d s i gni f i c a nt l y t o 0.4752 pe r 10000 pi xe l s . p
= 0.0350
2 5
D H M m od u l at e s t h e m or p h ol ogi c al c h an ge s i n as t r oc yt e af t e r s oc i al i s ol at i on
F i gu r e 8. D H M m odul a t e s t he m or phol ogi c a l c ha nge s i n a s t r oc yt e s a f t e r s oc i a l i s ol a t i on. T he s ke l e t on of t he a s t r oc yt e w a s
a na l yz e d by F i j i S of t w a r e . A . A ve r a ge br a nc h num be r of e a c h s i ngl e a s t r oc yt e a nd a ve r a ge a s t r oc yt e br a nc h l e ngt h i n C or nu
A m m o n i s 1 ( C A 1 ) B . A v e r a g e b r a n c h n u m b e r o f e a c h s i n g l e a s t r o c y t e a n d a v e r a g e a s t r o c y t e b r a n c h l e n g t h i n D e n t a t e G y r u s ( D G )
r e gi on. n = 3- 4 e a c h gr oup. G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; I + V : s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ; I + D :
s oc i a l i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt . U s e O ne - W a y A N O V A f ol l ow e d by m ul t i pl e c om pa r i s ons t o a na l ys i s .
D a t a w e r e a l l s how n a s m e a n ± S E M . * p < 0.05
N e ur ol ogi c a l di s e a s e s not onl y l e a d t o a n i nc r e a s e i n t he num be r of a s t r oc yt e s , but a l s o
of t e n l e a d t o c ha nge s i n t he i r m or phol ogy. U nde r t he i nf l ue nc e of e xt e r na l pr e s s ur e , t he ne r ve
s yna ps e s i n t he br a i n a r e of t e n t he m os t vul ne r a bl e t o da m a ge w i t h s hor t e ne d l e ngt h a nd r e duc e d
2 6
num be r s . I n or de r t o m a i nt a i n t he f unc t i on a nd s t r uc t ur e of s yna ps e s , a s t r oc yt e s w i l l e xhi bi t a n
i nc r e a s e d t r e nd i n t he num be r a nd l e ngt h of br a nc he s . S ke l e t on a na l ys i s i s one of t he m e t hods t o
qua nt i f y t he m or phol ogi c a l c ha nge i n a s t r oc yt e s . W e c a l c ul a t e d t he a ve r a ge br a nc h num be r of
e a c h s i ngl e a s t r oc yt e a nd a ve r a ge br a nc h l e ngt h i n C A 1 a nd D G a r e a by us i ng t hi s m e t hod.
F or t he C A 1 r e gi on, w e c a n obs e r ve t ha t t he a ve r a ge br a nc h num be r w a s s i gni f i c a nt l y
i nc r e a s e d a f t e r 4 w e e ks of i s ol a t i on. ( I s ol a t i on gr oup 22.77 ± 1.469 vs c ont r ol gr oup 17.36 ±
0.674) p = 0.0214 by us i ng D unne t t ’ s m ul t i pl e c om pa r i s ons a f t e r O ne - W a y A N O V A . A f t e r
t r e a t m e nt w i t h D H M , t he br a nc h num be r f or e a c h a s t r oc yt e i n t he I + D gr oup w a s r e duc e d t o
( 17.45 ± 0.984) s a m e l e ve l a s gr oupe d m i c e . I n t he D G r e gi on, w e c a n obs e r ve t he s a m e c ha nge .
C om pa r e d w i t h t he c ont r ol gr oup ( 17.86 ± 0.206) , t he br a nc h num be r i n i s ol a t i on m i c e i nc r e a s e d
t o 19.71 ± 0.362 w hi l e t hi s a s t r oc yt e a c t i va t i on c a n be r e l i e ve d by D H M . H ow e ve r , t he r e w a s no
di f f e r e nc e i n t he a ve r a ge br a nc h l e ngt h a f t e r i s ol a t i on or D H M t r e a t m e nt bot h i n t he C A 1 a nd
D G r e gi ons . T he s e r e s ul t s i ndi c a t e t ha t t he m or phol ogy of a s t r oc yt e s w a s c ha nge d m a i nl y
t hr ough t he i nc r e a s e i n t he num be r of br a nc he s unde r t he s oc i a l i s ol a t i on s t i m ul a t i on. A nd D H M
c a n pr e ve nt t he gr ow t h of e xt r a a s t r oc yt e br a nc he s .
2 7
C h a p t e r 3 . D i s c u s s i o n
A bout one - t hi r d of t he gl oba l popul a t i on w i l l be t r oubl e d by a nxi e t y di s or de r s i n t he i r
l i f e t i m e . A nxi e t y di s or de r s c a n of t e n a c c om pa ny ot he r ne ur ol ogi c a l di s e a s e s a nd l ong- t e r m
a nxi e t y i s a l s o a m a j or c a us e of A l z he i m e r ’ s di s e a s e . B e c a us e of C O V I D - 19, pe opl e s pe nd m or e
t i m e s t a yi ng a t hom e w hi c h i s c ons i de r e d a s a m a j or c a us e of a nxi e t y di s or de r s . S o i n c ur r e nt
s t udy, w e us e d a s oc i a l i s ol a t i on m e t hod t o e s t a bl i s h a m i c e a nxi e t y m ode l , w hi c h s how a nxi e t y
be ha vi or a l c ha nge s . The pr i m a r y a i m of t hi s s t udy i s t o unde r s t a nd unde r l yi ng m e c ha ni s m of
a nxi e t y a nd r e l a t e d ne ur o- i m m une s ys t e m a l t e r a t i on i nduc e d by s oc i a l i s ol a t i on. B a s e d on our
t e a m ' s pr e vi ous r e s e a r c h, D H M ha s a l r e a dy s how n t he a nxi ol yt i c pr ope r t y i n a ge d t r a ns ge ni c A D
m i c e ( T G 2576 a nd T G - S w D I ) a nd s oc i a l i s ol a t i on m i c e .[ 33] [ 34] I n t hi s s t udy, w e f ound t ha t
s oc i a l i s ol a t i on c a n l e a d t o a n i nc r e a s e d a nxi e t y- l i ke be ha vi or i n m i c e , a r e duc t i on i n B D N F l e ve l
a nd ove r a c t i vi t y i n t he num be r a nd m or phol ogy of a s t r oc yt e s . I n c ont r a s t , D H M c oul d i m pr ove
t he a nxi e t y- l i ke be ha vi or , r e s t or e t he r e duc e d B D N F pr ot e i n e xpr e s s i on a nd r e ve r s e t he
ove r a c t i vi t y i n a s t r oc yt e s . O ur r e s ul t s i ndi c a t e t ha t D H M i s a pot e nt i a l c a ndi da t e f or t r e a t i ng
a nxi e t y di s or de r s .
T o e va l ua t e t he hypot he s i s t ha t D H M a m e l i or a t e s a nxi e t y a nd ge t a be t t e r unde r s t a ndi ng of
i t s m e c ha ni s m , w e i nduc e d a m i c e a nxi e t y m ode l by s oc i a l i s ol a t i on. T o a s s e s s a nxi e t y- l i ke
be ha vi or , e l e va t e d- pl us m a z e a nd ope n f i e l d t e s t s w e r e c onduc t e d i n our s t udy. B e c a us e m i c e a r e
noc t ur na l a ni m a l s , a l l t he be ha vi or t e s t s w e r e s t a r t e d i n t he da r k pha s e t o m i ni m i z e t he i nf l ue nc e
of c i r c a di a n c yc l e . W e f ound t ha t s i ngl e - hous e d m i c e e xhi bi t e d a nxi e t y- l i ke be ha vi or t ha t
s how e d r e duc e d m obi l i t y, r e duc e d e xpl or a t or y be ha vi or , a nd a gr e a t e r t e nde nc y t o s t a y i n c l os e d
e nvi r onm e nt s . H ow e ve r , i s ol a t e d m i c e t r e a t e d w i t h D H M s how e d a s i gni f i c a nt i m pr ove m e nt i n
a nxi e t y- l i ke be ha vi or w hi c h i s s i m i l a r t o t he c ont r ol gr oup.
2 8
B D N F pl a ys a n i m por t a nt r ol e i n pr ovi di ng nut r i t i on f or s yna ps e gr ow t h a nd m a i nt a i ni ng
i t s nor m a l f unc t i on. M a ny pr e c l i ni c a l a nd c l i ni c a l s t udi e s ha ve a l r e a dy s how n t he a s s oc i a t i on
b e t w e e n d y s r e g u l a t i o n o f B D N F a n d c h r o n i c p s y c h o l o g i c a l d i s o r d e r s i n c l u d i n g a n x i e t y d i s o r d e r s .
I n our e xpe r i m e nt , w e obs e r ve d a s i gni f i c a nt de c r e a s e i n B D N F pr ot e i n e xpr e s s i on i n s oc i a l
i s ol a t i on m i c e . I n J ova novi c ’ s r e s e a r c h, he poi nt e d out t ha t t he l os s of s yna ps e r e l i e s on
B D N F - de pe nde nt r e duc t i on of G A B A r e c e pt or a nd de c r e a s e d i nhi bi t or y G A B A e r gi c
ne ur ot r a ns m i s s i on.[ 35] I n m a ny ot he r ne ur ol ogi c a l di s e a s e s , e s pe c i a l l y A l z he i m e r di s e a s e , t he
de c r e a s e of ne ur on c e l l s a nd t he l os s of s yna pt i c f unc t i on a r e c ons i de r e d a s t he t w o m os t obvi ous
pa t hol ogi c a l f e a t ur e s . A s t r oc yt e s a r e de s c r i be d a s pl a yi ng a s uppor t i ve r ol e i n t he t r a ns m i s s i on
a nd pl a s t i c i t y of s yna ps e s , w hi c h i s c a l l e d t r i pa r t i t e s yna ps e .[ 36] T he r e f or e , w he n s yna ps e s a r e
s hor t e ne d a nd l os e f unc t i on due t o di s e a s e , a s t r oc yt e s a r e s t i m ul a t e d t o i nc r e a s e t he i r br a nc h t o
m a i nt a i n t he nor m a l f unc t i on of s yna ps e s . T o ve r i f y our hypot he s i s , w e us e d F i j i t o a na l yz e t he
m or phol ogy a nd t he num be r of a s t r oc yt e s . W e f ound t ha t i n t he s oc i a l i s ol a t i on gr oup, a s t r oc yt e s
i nc r e a s e d t he i r de ns i t y a nd m or phol ogy c om pl e xi t y. H ow e ve r , D H M e xhi bi t e d i t s a nxi ol yt i c
pr ope r t y by m a i nt a i ni ng t he nor m a l l e ve l of B D N F a nd pr e ve nt i ng t he a s t r oc yt e s f r om a c t i va t i on.
I n our t e a m ’ s pr e vi ous s t udy, w e f ound t ha t D H M a l s o ha s s om e a dva nt a ge s i n a nxi ol yt i c
pr ope r t y c om pa r e d w i t h di a z e pa m w hi c h i s c ons i de r e d a s t he f i r s t - l i ne t r e a t m e nt f or a nxi e t y
di s or de r .[ 14] I n t he s a m e be ha vi or t e s t s , di a z e pa m t r e a t m e nt s how e d l i m i t e d e f f e c t s i n t he
c ha nge of be ha vi or a l pa r a m e t e r s c om pa r e d w i t h D H M . G e phyr i n i s c ons i de r e d a s c a f f ol di ng
pr ot e i n f ound m a i nl y i n i nhi bi t or y s yna ps e s . I t ha s be e n de t e r m i ne d t o i nt e r a c t di r e c t l y w i t h
gl yc i ne r e c e pt or s , t he m a j or r e c e pt or pr ot e i n a t i nhi bi t or y s yna ps e s i n t he s pi na l c or d a nd
br a i ns t e m , but i t a l s o i nt e r a c t s di r e c t l y w i t h c e r t a i n ga m m a - a m i nobut yr i c a c i d A r e c e pt or s
( G A B A - A ) r e c e pt or s i n t he r e s t of t he br a i n t o m e di a t e i nhi bi t i on i n t he br a i n. I n t he W e s t e r n
2 9
B l ot e xpe r i m e nt , t he r e i s a s i gni f i c a nt di f f e r e nc e be t w e e n D H M gr oup a nd di a z e pa m gr oup a nd
t he nor m a l i z e d ge phyr i n de ns i t y i s m uc h l ow e r i n di a z e pa m t r e a t m e nt gr oup.
T hr ough t he B D N F - T r kB s i gna l i ng pa t hw a y, s oc i a l i s ol a t i on not onl y c a us e s t he a c t i va t i on
of a s t r oc yt e s , but a l s o c a n s t i m ul a t e t he a c t i va t i on of m i c r ogl i a .[ 37] A l t hough i n t hi s s t udy, w e
di d not f i nd a ny di f f e r e nc e i n N F - κ B pa t hw a y, ne ur oi nf l a m m a t i on s t i l l i s a hot t r e a t m e nt s t r a t e gy
f or ps yc hi a t r i c pa t i e nt s .[ 38] M i c r ogl i a w hi c h ha s be e n w i de l y r e c ogni z e d a s a n i m por t a nt i nde x
of ne ur oi nf l a m m a t i on i s a ki nd of i nna t e i m m une c e l l i n t he c e nt r a l ne r vous s ys t e m . B a s e d on t he
J i ng ’ s r e s e a r c h, D H M c a n a l l e vi a t e L P S - i nduc e d ne ur oi nf l a m m a t i on a nd ove r a c t i va t e d
m i c r ogl i a .[ 39] W e ha ve a l r e a dy us e d t he f r e e - f l oa t i ng m e t hod t o s t a i n m i c r ogl i a by us i ng i ba - 1
a nt i body a nd t a ke n s e ve r a l i m a ge s unde r f l uor e s c e nc e m i c r os c ope ( F i gu r e 8) . H ow e ve r , t he
m or phol ogy of m i c r ogl i a c oul d not be di s t i ngui s he d ve r y c l e a r l y be c a us e of t he s t r ong
ba c kgr ound s i gna l . I n our f ur t he r s t udy, w e w i l l f oc us on t he m or phol ogy of m i c r ogl i a t o ha ve a
m or e c om pr e he ns i ve unde r s t a ndi ng of t he t r e a t m e nt m e c ha ni s m of D H M . H e nc e , D H M ha s
br oa d c l i ni c a l a ppl i c a t i on pr os pe c t s i n l ong- t e r m t he r a py f or a nxi e t y di s or de r s a s w e l l a s
r e duc i ng t he pot e nt i a l s i de e f f e c t s .
3 0
F i gu r e 9. R e pr e s e nt a t i ve m i c r ogl i a i m a ge s t a ke n by B i o- T e k C yt a t i on 5 C e l l I m a gi ng M ul t i - M ode R e a de r a t a m a gni f i c a t i on of
20. I m a ge s s how I ba - 1 s t a i ne d m i c r ogl i a a nd D A P I s t a i ne d nuc l e us i n m i c e hi ppoc a m pus C or nu A m m oni s 1 ( C A 1) r e gi on. S c a l e
ba r e qua l s 100um . G + V : gr oupe d m i c e w i t h ve hi c l e t r e a t m e nt ; I + V : s oc i a l i s ol a t i on m i c e w i t h ve hi c l e t r e a t m e nt ; I + D : s oc i a l
i s ol a t i on m i c e w i t h 2 m g/ kg D H M t r e a t m e nt .
3 1
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3 3
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doi : 10.1515/ m e d- 2019- 0083

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Creator Zhang, Jifeng (author)

Core Title Social isolation anxiety induced synaptic failure restored by dihydromyricetin

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School School of Pharmacy

Degree Master of Science

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2021-08

Publication Date 07/26/2021

Defense Date 07/23/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag anxiety,dihydromyricetin,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,synaptic failure

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Advisor Jing, Liang (committee chair), Beringer, Paul (committee member), Davies, Daryl L. (committee member)

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Abstract (if available)

Abstract Anxiety disorders are a series of chronic mental diseases that affect many people all over the world, which are mainly characterized by significant and uncontrollable feelings of anxiety and depressed. During the COVID-19, people’s lives have been greatly affected. Long-term quarantine at home and lack of social activities have led to getting anxiety disorders more easily. Dihydromyricetin (DHM), a kind of bioactive flavonoid, shows the effects on decreasing alcohol usage, reducing anxiety-related behaviors and repairing cognition impairment in our team's previous studies. In current study, using a social isolation mice model established in our lab, we found that isolated animals showed increased anxiety levels in elevated plus maze and open field behavior assays. We also found a decreased brain-derived neurotrophic factor (BDNF) level as well as activated astrocytes, glial cells which stabilize synaptic connection and maintain synaptic function. While DHM can restore the BDNF concentration to a normal level and also alleviates the activation of astrocyte by decreasing the number of astrocyte and reducing the branch number. Our study suggested that short term of social isolation could induces astrocyte overactivation and BDNF reduction, both contribute to synaptic functions. DHM can restore the synapse dysfunction and reduce anxiety level, suggesting DHM can be considered as a novel candidate for treating anxiety disorders.