Experimental and modeling studies of motor network excitability of neonatal rat spinal cord in vitro

Experimental and modeling studies of motor network

excitability of neonatal rat spinal cord

in vitro

!

"

!

Table of contents

NOTE 4

ACKNOWLEDGEMENTS 5

ABSTRACT 6

INTRODUCTION 8

1. Anatomical overview of the spinal cord 8

% % $ & ' % ( ) * + & , % - " . / %0 % 1 2 %% % 3 2 %( % 4 " 5 %-% 6 7 . %4 % ' %6 % , & %'

2. Spinal reflexes. Development in rats 19

( % 8 8 (0

( ( 8 (%

( - $ &

(-( 1 8 & (1

3. Locomotion and locomotor related spinal networks 25

- % " & 9 (3

- ( ) : " " (6

- - " ('

- 1 7 8 # (,

Aims of the present study 32

METHODS 33

1. Tissue preparation and drugs 33

% % --% ( 7 8 --% - 7 . : 8 -1 % 1 ;8 < -3 2. Recording techniques 36 ( % ;8 " -4 ( ( -6 ( - ; -' ( 1 7 : -, ( 3 ;8 -, ( 4 = 1% ( 6 1(

3. Data analysis 44 - % 11 - ( ) 11 - - 11 4. Molecular biology 45 1 % > 13 1 ( 7 : 7 13

5. Modeling of motoneuron structure 47

3 % 2 16 3 ( 2 8 16 3 - 2 8 16 3 1 2 1' 3 3 1' 3 4 " 1' RESULTS 49 1 Experimental results 49 % % ; " / " 1, % ( ; " 3% % - 7 " / 31 % 1 2 36 % 3 : 4-% 4 " ? 7 43 % 6 ? 7 46 2 Modeling results 70 ( % 60 ( ( 8 6% ( - 8 6( ( 1 63 ( 3 2 8 6' ( 4 ;8 " '0 ( 6 2 ? 7 / " '% DISCUSSION 86 " 8 #@ _{/ '4} . / " " / '' ? 7 : _'' A . ? 7 ; : _{. B ',} ? 7 8 ,% # /B ,% ? ,( " ,( 2 " ,-" 8 ? 7 ,3 REFERENCES 98

Note

< / . ! ;< < *(001+ " " < %(13:%(31 ;< < *(003+ 2 " " %-,:%3, < < ;< 9 *(004+ ! . " -(-:--1 < 9 2< *(004+ " 8 * + . / . 8 < < . < 8 7 : 7< > : . 2 9 7 . ; < . : <

Acknowledgements

. / / " A . . / . / / 2 9 . / . . / / 9 " 2 . " < . . / / ; 5 / < . / . / "

Abstract

" . / 8 < < 8 " < . : " " " " #@ *# + 8 * %,,-C 2 " < %,,'+ < . / < # D * %,,4C < (003+ E < . / " < " . / . 8 *# F E < (00-+ . " " . : @_:#@ 5 . / *7 && < (00(+< . . . " # 8 $ . # / / *E < (001+ . / 8 < . " . / 8 E / * ? 7+< " " : _< 8 8 . 8 : 9 E * +< ? 7 * & ? 7 :%6(+ & < / " . " " 9 E 7 : 7 > . ? 7

8 ? 7 : _{8 <} 8 . . 9 E " ? 7 / . 8 " -. . . / . " : " " 8 & ? 7 " & . 9 E G :

Introduction

. / " " " D " < 8< " . & 8 / *# < (000C 2 < (00%C 2 < (001+ " " " " < . . . . * < %,04C < (003 +< . / :. 8 *? & < %,'3C # F H < %,'6+ * 9+ 8 . / . * / + *E < (00(C # F E < (00-C < (001C # < (004+

1. Anatomical overview of the spinal cord

1.1 Organization of the neonatal rat spinal cord

" . * + " & " ! " * " < +< * . " < +< * 8 " < 8 + * < + " . 8 < 8 < < . " " /< " C / . 8 /. C " " < . " < < 3 *)3+

D < " . / < < < . . " / . 8 & " . / : * " . &< (00(+ " . 8 * / F $ " / < %,64+ . &

1.2 Laminar (cytoarchitectonic) organization of the rat spinal cord

" I < * + . *. + : / < . . < . " < 7 8 *%,3(+ *2 # F # A7< %,,3 C 7 " :2 < %,,4+ *? & < %,,%C #5 < %,,1C ) " < (00(C # F E < (00-+ 2 *%,'1+ 8 7 8 J . & . 2 " < & . . < ? % . )3 & < . " < < < "

1.3 Spinal motoneuron development up to the second postnatal

week

< %( *;%(+ < ;%1< . . / 3 * ;%% ;%4C F < %,6(C %,61+ ) / < : : . . ;%3 * %+< . " *$ < %,'4C " . $ < %,,%+ A < < . " " < 5 " " " . * .+ & 8 *)K F < %,,(C > < %,'(+< "

. " ) / . < " < " " ? < . * . ' -0+ . > 9 *%,,(+< * %:,+ / 8 " " // *%,,1+ < 6< . : * + 8 < & (0 * & + 5 . & (0< %4 < . . / < " 2 " 8 8 *= < (000+

1.4 Motoneuronal pools

# . 5 8 < & 8 " 8 " 8 *A7 + * : F < %,'-C F < %,'-C / < %,'6C # < %,,3C # F < (000+ E & . * " +< & " 8 5 "

8 : F *%,'-+

1.5 Motor units

< 8 < " " *=7+ ? 8 . ;%-:%1 * F < %,6(+ : ;%1 * < %,'%+ ;%'< < " . . / * < %,'%+ *%,'-+ )1" D %'1( %34'< " . . D * : F < %,'-+ 2 8 . * 8 8 / 7 . +< " A . " < " " * F ) < %,,,+ " < . / 8 < . . " < " < / ? ( * < (003 + " " " . 2 . * " . %,'6C 9 " < (000C L < (000C # F < (00(C F E < (001+ . . 8 * " . # F < (00(+ " &

1.6 Afferent nerves and afferent projections to the rat spinal cord

; " * 7+ " * " + 7 " < 8 * 79+ 79 5 . ;%3:;%6 *? & < %,,%C 2 # F # A7< %,,3 + ) C : < *;%,:(%+ * < %,,(C ? & F L < %,,,C 2 # F # A7< %,,3 + E " " . " " " . / . . " " " " *? & < %,'3+ " * 79 5 + / / ;%- . *2 # F # A7< %,,3 + " 7 * % + < . * % + " < 5 . " . ) *%,''+ . " := 5 " " < = . 5 A . " < β = 8 * < %,,(C ? & < %,,1C ? & F L < %,,,+ . . . " . / *? & < %,,1+ 2 " < 8 . : < . & 5 < . < 7 8 * : < %,,3+

! "

#$ #$ #$ #$ % $ &&' type of fibre diameter, µm myelination conduction

velocity, m/s general function

Ia 12-20 yes 70-120 muscle spindle _{primary endings}

Ib 11-19 yes 66-114 Golgi tendon organs

II 5-12 yes 20-50

touch, kinesthesia, muscle spindle secondary endings

III 1-5 yes 4-20

pain, crude touch, pressure,

temperature

IV 0.1-2 no 0.2-3 pain, pressure, _{touch,temperature}

$ type of fibre diameter, µm myelination conduction velocity, m/s general function A-α 13-22 yes 70-120 alpha-motoneurons, muscle spindle primary endings, Golgi tendon organs, touch

A-β 8-13 yes 40-70

touch, kinesthesia, muscle spindle secondary endings

A-γ 4-8 yes 15-40 touch, pressure, gamma-_motoneurons

A-δ 1-4 yes 5-15 pain, crude touch, _{pressure, temperature}

B 1-3 yes 3-14 preganglionic autonomic

C 0.1-1 no 0.2-2

pain, touch, pressure, temperature, postganglionic autonomic 5 M & & < " " 8 5 5 "

*7 " :2 < %,,4+ " . * + . *%,,(+ " < . < < " 5 < " . . . " . " J 8 ? . 5 < . " < *;%6+ *. +< & )3* =:= C < %,,(+ . ;%6 3:;%, 3 *# F H < %,'6+

1.7 Renshaw cells

< 7 . * " . A < (004+ . ! < 7 . . 8 . 7 / . * F < (00(+C < *A < (004+ 7 . " * = C " & < %,,,+C . " " 8 5 " * " & < %,,,+ 7 *2 < (003C < (003+ " . 7 . ! < < " 2 2 G/ 7 .

( ( ( ( % $ ) $ % & ' α γ ' α ' % & ' % & ' ' $ ' ( ' )( * ' ' + ,--. < 7 . < : " *2 < (00(C A < (004+ . . . " . " . " * " . A < (004C ? -+

1.8 Commissural interneurons

* + . " : * .C " . E < (00(C # F E < (00-C # < (00-C # < (004+ & * : F < %,,(C : F < %,,1C E < (00(+

; " : *%,,(+ : " *%' ;%,+ . < & " E ;%3< 8 . 8 & " C < . " < # " & 8 5 *; < %,,,C F < (000C " . E < (00(+ ) / < < " & A . " < & 8 8 5 . * + . * + 8 " . " & < . . & *; < %,,,+ 5 8 " * + * + E * C %6 N + 5 * C (0 N + *E < (00(+ E *(00-+ . . ? < . . 8

1.9 Summary of the spinal cord organization

&

: 5 . " & & ! %< 5 D & C (< 8 " " C -< & C 1< 8 . " . : < : : 7 . C 3< / . 8 / " < 8

2. Spinal reflexes. Development in rats

8 " > " < " & < : " 5 : 8 " " " . ! . " . * &/ < (000C < (003 < + A . " < 8 < & * &/ < (000+ 8 . :. I . 8 8 < . / " : / " 8 : < : . < :.

2.1 Stretch reflex and monosynaptic reflexes

8* 8 + 8 8 . 5 / 8 8 8 5 . / ) 8 %,(1 %,(3 > / 8 *D + . < " " " > " . < " . . 8 . C < < 8 . 8 " D " A: 8< A * +< %,%0 " / 8 " : " 8 . " # F H *%,'3+ : ! %< 8 ;%, 3< . * " +C (< 8 " / * + C -< 8 *; + ; < & 8 / : : . 8 < " < " " " * # F H < %,'3+ ) ": " *%,,(+ " * 4: '+ 9 E : " * : / + " "

# F H *%,'3+< " " " " " 8 " * < %,6,C ? & < %,'6C # F H < %,'6+ 8 )1 =7 ;%' 3 . )1 7 *# F H < %,'6+ * " / 7+ ;%4 3:;%6 3 & < " * < %,6,+ . =7 8 *? & < %,'6+ / . < . 8 8 8 8 8 / . " " " . %,14< ) O J! . < . " *? (+ : " O J * + < . * C ? - < + 9 < . . < " < < .

2.2 Polysynaptic reflexes

8 8 " " " . " < 8 8< " 8< . . 8 8 . / 8 C <

8 C " < 8 < < " 8 < . =7 7 < " / *;%3 3+ 8 & =7 . & " . / * < %,6,+ . *;%6+ " / . . 8 * ? & %,'3+ 2 " < < / " / 8 *? & F 9 < %,'1+ . . 8 < : " / 8 / %0: %% *? & F 9 < %,'1+ < " . " .: : ! < : " / 8 : *? & F L < %,,,+ " " " " & " . 2 ? & 7 . " . *;%,+ " " *;%6+ *? & < %,,%+ " " * + " * 0: -+< . / " * & + E %1:%3 3%N " < 1,N < . " 8 " 8 . / < *? & < %,'3+ < " *? & < %,'3+ ) / 5 " < " : < " " . " : D 2 " . 2 2#'0%<

" : " / <

. : " / "

& : ! . . :

<

*E < (00(+

2.3 Organization of inputs to spinal interneuron population and

reconfiguration of spinal interneuronal system

) =:= *P P< P " P< P P P .P+ & * & < (00-+ 5 . * & < (00-+ 5 * " C : F < %,,(C : F < %,,1C ; < %,,,+ 7 . < 8 < * " + ? - * + . " / 8 < 7 . " 8 - : : 7 . " *> < %,,4C A < (004+ ; *(00%+ L / . / *(00%+ " " . & < : > < ; 8 & . < α: γ: "

< 7 . * ? - O J 7 . + 2 . & . ! . " < . 7 . 8 . . . / L / . / *(00%+ " . / * ? % L / . / < (00%+ / *# F ) ": "< %,,'C = < %,,,C 7 < %,,,+ $ ! " " " < < < . / < .

2.4 Summary of spinal reflexes and functional organization of

spinal cord

. * < C " . ) . F ) . < (000+ 2 " < " < . / . < . / : ; < " ? < " 8 > & / < 8 8 * = " / < (004+ A . " < 8 < . " " $ 5 " .

& < / 8

8 . . " /

< .

/ .

* 9+

3. Locomotion and locomotor related spinal networks

/ $ " / *%,64+< " . < " < D . " . . " D 5 " 8 < * < 5 " + " " " . ! . . " /. < . < . " .

3.1 Development and localization of locomotor CPG in rats

) . . ! " " " . 5 " < D * + ;8 * " . / F $ " / < %,64+ < . D . / " " < < D * " . # F E < (00-C < (001C < (004C # < (004+ 2 8< < " . /

* 9+ 40 :60 . " " / '0 " 9 " < * " . < (000C &< (00-+ > / . 9 " *$ " / < %,,,+< 9 < . " < * < (001C # < (004C < (004+ $ D 9 8 . J " " . 9 " 8 " G < . =7 . " *)(G)- " 8 < . )3 " 8 + : " < / : : : * 2 +< < 3: 8 * 3:A +< * F # < (000+ : / " " / D 7 *2 < (00% + " 8 #@ _{*E < %,,'+} < " " " / 2 G 3:A *;%1 3:;%4 3+< . < ;%' 3 =7 )(G)-< . )3 =7 . )(G)- =7 . . . " " " 5 * " + * " 8 + ? 9 " . # *(000+ & 9 " . # *(004+ E < " *

= < = M+ " < < . " < . " : < 9 & < : & " " 5 " < . * / F $ " / < %,64+

3.2 Left-right coordination during fictive locomotion and its

development

: " : . = * " 8 5 + : * " . E < (00(C E F # < (00-C # < (004+ *%,,4+ & =7 . . " ;%1 3 ;%3 3 $ ;%6 3 / . 9 E " 8 * < %,,4C # F < %,,'+ < " 2 3:A C : ;%' 3 " : ;(0 3 . 8 9 E : * 8 C / < (00(+ " . / 8 " *# F < %,,'+

3.3 Disinhibited activity in the isolated spinal cord of the neonatal

rat

E / -: %- . : 8 " " / " 8 * D Q( < Q6 +< . *E < %,,4 C E < %,,6+ < . / ; & . : *E < %,,4 < C +< < 8 < / 9 E < . " " *E < %,,4 C + " / #@ _{* F < (001<} (004+ " . / < D < < ? < " : " 0 = " ? . / / " : " @ 8 " : " (@ _/ (@_*E < %,,4 + 8 8 8 . / 8 < : & 8 8 " " * : + 8 *E < %,,4 C E F < %,,,+ 8 " . 8 . : /

" " 3:A G 2 < *3:A G 2 + ) " . . / D *E %,,4 C #5 F # < %,,4+ < . " " . . *E %,,4 +< )1 )3 =7 " 8 * + 8 * + 2 * F # < (000+

3.4 Role of cell metabolism in the control of neuronal excitability.

K

ATP

channels

& " @ _{* +} * < (001+< $ & / " < . 5 " *9 < + > 8 " " " < G# / *E < %,,6C 7 && < (00(+< . @ " < % /< . / *7 && < (00(+ < =7 < < < R%( " . . *7 && < (00(+< " :

" 8 " 8 : " " / * / < (000C / < (00%+ " : < . " : " < 8 " " #@ _{*# +< / . / .} 8 * " . E < (001C 2 < (001C H F / < (003C < (004+ " . G " # " β: > < < # & (@ _< (@ _{" #} & < . *? 1 C < (003C < (004+ # D . $ < # 4< # C < I7 * +< : * E + . # 4 < . " . I7 ? 1 . & # . # # 4 ( #@ _I7% : " * :E F E < %,,,+ # 4 ( 8 * & < (003+< 8 " / .

# *+#, β) / 012031 401 5 !6 !6 _{' 401 01} 5 46 4 . 7 )3 8(9 8), )! 81 ( !"". ;8 " " " # " # " . * < %,,4C A < (00%+< " : . *30 S2+ *300 S2+ . / # < " I7% * " . E < (001+ " 5 " > 8 D

Aims of the present study

. " . / : @_:#@ ₅ . / *7 && < (00(+< . . " # 8 $ # / / *E < (001+ . / 8 . " . / 8 ? < . " . 9 E " " < - < " . / < " 8

Methods

1. Tissue preparation and drugs

1.1 Spinal cord tissue preparation

< . " < . > *1 ' < 1: '+ . & . 5 *0 ( %0N . G" + . . 8< . . " . " . " 8 " " / . " . . *1 + 8 *,3N $(: 3N $(+ * ?C .+ 8 . " ? . 8 . 2 . " " < . 7 =7 . 7 . " ? . ? / < ? * .+< . 8 *. + / = . : 8 ? / " (00 -00 S . % -( 8 D %

1.2 Recording chamber for the isolated spinal cord experiments

< . . *" . +

. . ? . . . . " 8 ? < " < 8 ? . . " . . . . " ? 8 . . 3 6 G . " 8 . ? .

-1.3 Recording chamber for the whole cell-patch experiments and

identification of the cells

. 8 : . *(:- G + . 8 ? * .+ . . . ! : " 8 * 3:%0 D +< " < ? < :. . . . *- : < ) 2 < 9 + 3:4 2Ω . . 1: *9 E C 300 S2 8 +< 8 (0:30 S . < . . . ! " / / J . *> < < ?)< I +C < %0 (0 *1:' + 2 . " *R (0 S + " * MC / < %,,0+ . *T%3 S + " * M+

1.4 Extracellular, intracellular solutions and drugs

! . * 2+! %%-< # 1 3< 2 (6A($ %< ((< A( $1 %< A $- (3 %%< . ,3N $(: 3N $(< A 6 1 8 . "# $ . . ( 2 #2 $1 ? * + (0 2 UM:-%1 . & @_{: / &} %# $ . . ( 2 ( $1 (0 2 UM:-%1 < * 2+! %-0< # -< A( $1% 3< (%< 2 (3< A $-(3< %% * A 6 1 5 . $AC (,0:-%0 %_{+ 8} * 2+! %-0< # -< A( $1 % 3< (% 3< 2 (%< A $-(3< %% * A 6 1 5 . $AC (,0: -%0 %₊ &# $ . . * 2+ # %-0< 3< 2 ( (< ( 0 %< A; ; %0< ;9 3< :2 (< 9 : %< UM:-%1 0 3 * A 6 ( . #$AC ('0 -00 %_{+ ? < 2} ': : 2 *100µ2+ . ' # ($ $ ($

. . * 2+ : %0,< '< 2 ( (< '< A; ; %0< ;9 %< :2 1< -9 0 -< UM:-%1 0 3 * A 6 ( . $AC ('0 -00 %₊ )* ? 8 . < < 1: 8 D :(: 8 */ +< 9 E < < : < : *UM:-%1+< :(<( : 8 * + * : <2 < + . ? / *E < . & + 9 . * E < ; " < 2 < I + ? 7 :%6( *-:V*-: + W:3:V*1: 8 + W:(: 8 :1: & + . * < 9 + / * %0- ₊ . . & D *T % " + . " / ? . " 8 * 2 $+ . 2 $ . X0 0( N ? > 8 < / . 7 *? A :) 7 ) < E < . & + E . * : < ) < 2$< I + -:' N : 9; " 8 . " * " 7 )< 9 2 < + : ? 7 . * ) ) < L < +

2. Recording techniques

2.1 Extracellular ventral root recordings

= . .

; .

& . * + . =7 * +

" . > . < " . . " ; . " . ? " G : *A " < I#+ .: 2 30 *> < ?)< I )+ < . : 9 < & =A :

2.2 Intracellular recordings

. . . * " + " 0 %8 * 8 + * 8 ( < 8 + ; " . 30 ,0 2Ω #2 $1: . 40 %(0 2Ω ( $1: . . " < . * " D ( A&+ . ! " *%0 < 0 3 + < . < . 30 < *0 % + =7 * + > . 8 < " / *Q( + *? F > < %,'4+< . . . . < . < : 8 . < =7 *X( + : : " *? F > < %,'4+ $ . / .

; . * * + % 3:( 0 A& + 2 . 5 " *(0:30 +C . . & 5 5 E . . " " : < :" * :=+ . < " & *:0 % 0 , < . 0 %:0 ( " + & *0 %:0 , C " . + < . " 0 (:0 3 A& : " : 8 . " * ;= + D R( /A& R0 ' G = 8 ( . " . " (@ _UM:-%1 #@ _{/ / " : "} @ _< " E . ;= . 8 : _{" *;} :_{+ <} * + . " / *0 03 A&+ " / " ; : _. 8 " "

2.3 Electrical stimulation

2 . " " =7 . *% 3 + " . . ; . D *0 %:0 ( < 0 3:%0 = + . " %00 * ) < ; + 9

'' *9 2 < 2 < I +

=7 <

7 " ? <

" 7 . 8 * +<

=7

2.4 Recurrent post-synaptic potential recording

" 8 < . 7 . * + * + . * . . D +< #2 $1: $1: . " " :_{: < " ? <} " * + & < / . / / UM:-%1 / " / *2 < (00(+ / *- 2+< < . 8 8 *( S2+ *(0 S2+ . " 9 E * 2 < (00(+ ? 3 . . * + . =7 * + " :60 = " " :

2.5 Experimental configurations

. *)(: )3+ . 8 . " =7 7 < " 8 . .

-- . .+/+ : $ 0 1 1 ; ' . " . . " ;8 . =7 7 : 8 =7 " / . / *% 8 + *Y% 8 + 7 < " < . " 8 =7 ? 4 . 8 2 30 * %000C C :E + . . =7 " C 8 ( . 8 7 . ? 4 : =7 " *? 4 + . " . *? 4 + I < " < . & / < " " . D

' ' ' '!0) 1% 2 ' * ; ) < ; = 5 6,! 0 >" ; '< < ' ' < ' ; ' ' 3 ; < ' 3 ,* ?* '< * ( @ 8 A090 !" B) '< ' ' 9 < 3 ; ' ( 5 <

2.6 Viability of the spinal cord

< " . < *0 %:0 ( < 0 3:( =+ " 7 8 =7 " . < . Y%( . . 7 . / / "

2.7 Patch clamp recording

7 . " . : D *A %,'%+ . E < : < :. *A < 9 + . . : -:3 2Ω< . 9 < < . (:%0 9Ω * : + : < < . )G2 ; :6 *) 2 < 9 + . " 8 . :40< :43 = *= + *3:(3 2Ω+ . < . = D . . , ( . * 8 + . - /A& 3:%0 /A& > . " * / < %,,0+< . & : . = ? 6 . :" " 2 " < . :33 =< 44Z%% ? ''%Z-,0 2Ω * [10+ 8 : _{" *;} :₊ < 9 E . " / 9 E " * " C ? 6 + "

3 3 3 3 4 .+/+ 2; ' 5 * 8 A090 ' C A090 ' 5 A090 A090 A090 * / " & 9 E : " / < D . * Y10 C ? 6 + 9 E " / . " " . ; :_{* & < (003+} L . L * 8, (+ " ? : _{< 5 " D %0 = *} 2 < (00(+< . . : _{D %1 =} " . : "

3. Data analysis

3.1 Analysis of single cell recording

2 / " < / < / . " (0 / 2 . " . & . " . . " " * 0 %:0 ( + . := " < . " " & & . 5 & < . " / ? . : . 3 %0 = & * = +< G= . " * ! :'0 @(0 =< 1% 6 =G +

3.2 Linear statistic analysis

. 8 Z ; 2 < . 8 ; 2 E . < *# ": " + . : ? . . J $ * + . / : $= . : " " . [0 03

3.3 Analysis of disinhibited rhythm

<

" E . . < 3:%0 " * " 0 3:% D +< : : . /: : / "

4. Molecular biology

4.1 Western blot

. ? ' . : ? 7 * C %! -00+ %14':%1'0 ? 7 * \ %-34,+ . . A7 : 5 9 %!(000 *% +

4.2 RT-PCR

7 . . " & 8 & ? '

5. Modeling of motoneuron structure

5.1 Model of motoneuron soma

? 5 8 * + *%,,'+ . ;I7$ *A F " < %,,6+ A < " 8 " ;I7$ < D " . *-% 4%Z1 61 %4 0'Z( 1( " C " . %3N " +

5.2 Model of the axon

8 . *0 %< 0 % %0

8 /< 8 < " +

. . )

) / *%,,'+< /

8

5.3 Model of proximal dendrites

? < . . " . * // < %,,1C < %,,'+ . . & - * 8 . 0 + 8 .

5.4 Model of distal dendrites

) / 8 < . . 8 . " . " . . . Y,0 . < . " 7

5.5 Computer simulation and analysis

. 2 ) E " 6 0 7 %1 * 2 > / < + . 8 " " . 2 ) E . " 8 < . 8 " . " ) . . $ 4 % *$ ) < < 2 < I + J / . .

5.6 Simulation of electrical behavior of modeled motoneuron

; " . 8 8 " : " ;I7$ " > " . < " : / . " ;I7$ " * (3 S + ? . 8 @_{< # #} " *%,,3C (000+< . A / A 8 D * " < (000+

Results

1 Experimental results

1.1 Effect of glibenclamide on bicuculline evoked spontaneous

activity

" # 9 E : E *(0 S2+ . / 9 E *E < %,,4 C E < %,,4 + . / : " : #@ *L F < %,,6C L < (00%+ E " *? , + $ . / * E < %,,4 + " " / . & < < " : " " * ; + ; . & " " * =C ? , + . " *E < %,,4 + . =7 " & . =7 * [-C . + < # *30 S2C # < < %,,4+ < " . ; *? , . + ? , . & " / " * ['< X0 03+ < " < " ? , = < < " ; " = . C . " < " .

5 $ $ " ' !" 401 < 5 5 ' ' ' ' ' ' ; " . # / *300 S2C [-C . + > . " < *%00 S2+ 30 S2 30 S2 * [3C . + " . . ? < . " / " "

1.2 Effect of glibenclamide on the disinhibited activity

/ 9 E *E < %,,4 < + < 8 " . . " < . " # 8 ? < > . < * Q-0 + " =7 *? %0 + %0:%3 * . + ; " . & < . & * % 3 + . " *E < %,,4 < C E < %,,6+ 9 " " " " *? %0 . + . (0:-0 ? %0 : & " / " . " *300 S2< . C [-+ ? %% * 8 + * + * + 9 . *? %% +< . " D . 2 " < * + . * 8 ? %% + . * ? %% + < " & . . 300 S2 * . + A . " < " " 2 *3 S2+ 8 # *6 3 2 1 3 2+ ? %( . . &

& & & & 5 $ $ "6 " 0 #"B) ' '< 0 ' ' 0 ' ' ' ' ' $ 8

5 $ $ $ < ; 0 C 7 !+ 8 *0 $ $ $ * 4 ># ) ? # ) ( 5 4 5 D 4 ()30#B) 3 5 ()30

1.3 DR evoked responses

" . . #@ _{/ < . 8} " / 7 . " * . +< " " / . / *% 8 + *Y% 8 + 7 *2 < (00% + . 30 2 8 ? %-* + . (0 D . ? . / " * & + ? %- D 30 2 & . " . / < . < ) . *%:%0 2+ . " *3Z%0 N C Y0 03C [1+ ? %1 . =7 D *% A&+ *18 + 7 * /+ " * + . ? %1 < " < . / " < . 8 " . / . 8 *# / F E < %,,3+ < . =7 & " / 2 *(0 2+< 2 *(0 2+< 9 E *300 2+ *300 2+ ? %3 . & 2 2 * 8 + . < . & 9 E * 8 + . * ? %3 & +

( ( ( ( . $ 8 $ '< <,* ? * 3 ' ; !" #"E) ' ##" ' '< < * ( !" * C ' ; * F 3 #"E) GH I""# '

. $ " 9 ' ; C ,J5 ?* 3 < #"B) J 5 ' 5 GH8 I""# -- 0 $ "6 $ 8 $ $ < 5 ; #""E) A090 #"B) * 5 '< ()30 < #"B) J ; 5 F '< !# ()30 !" E) 0)10 !" E)

I""# A090 #""E) #""E)

1.4 Motoneuron electrophysiology

: 8 8 . . 8 . < . < . ; 7 . " / =7 *2 < (00(C 2 < (003+ . 8 ? %4 . < < < . ; .: 7 D ? %4 . ; < " < / . / *- 2+< *(0 2+ *% 2+ . /: : " I < . ? %6 < . & * " - 0Z0 , =C [%%C X0 006+ . *(,Z%0 NC [%%C X0 00-+ 8 & " / *? %6 + *(3Z3 N + . . *300 2C [%-+< # / . / . & < . & ? %6 ? %' . " G " " I D . . / / *- 4Z% % =C [%%C X0 00,+ " . .

' ' ' ' 0 $ 4,:, 5,:, 1 1 '< ; 8 D1 1 ; D1 1 ' 1 1 D1 1 I""#8GH & " *? %' + > < / " < . * [0 ',+ ? %' ( . / . < . . / *? %' + D ? %' * [4+ " . : " / 8 . & / . / / : #@ " : " /

3 33 3 9 6 " 9 $ G H, ;8 < 5 ( 5 HK ; < < "! 0 ; '< < ( ' 4 < K ) < !E) !"E) ;

0 $ ' 2 < ' ' 5 < ; ' G"H-8 GH 3 * F ' 0 ' >" ;8"! 0 = = < = , ' D F ' I""#8G.

#$ #$ #$

#$ 5 $ -& ;

parameter control glibenclamide P value

Vrest, mV -72 ± 1.4 -75 ± 1.3 0.007 *

Rin, M 21 ± 1.8 25 ± 2.4 0.03 *

overshoot, mV 15 ± 1.2 19 ± 1.1 0.009 *

spike rise time,

ms 1.3 ± 0.1 1.3 ± 0.1 0.98 inflection point, mV -47 ± 1.6 -46 ± 1.5 0.6 time to peak, ms 3.3 ± 0.2 3.5 ± 0.2 0.1 stimulus threshold, V 0.6 ± 0.1 0.6 ± 0.1 0.09 ) L D 8 G,,8; G 8 G 8M G < < E 8 . @_:#@ _{*E < %,,6C 7 && < (00(+<} " #@ _< @_:#@ / . ? %, . " / & *E < %,,6C 7 && < (00(+ . / :3% = * + . " & :63 = *. / " + D ? %, < " < & / @_:#@

. $ 8 $ " 9 7<_)*< 9 $ 6 >H ; <8 * 5 #, ; < 5 0 5 ># ; < ' < ' !" < " ; ?" !" 9 ' I""#8 G. * ' ' ' #" ; $ / : * ? 7C < %,,(C & < %,,,+< " " : _{" <} * F > < %,,,C F < (00-+< . 8 " *2 < %,,1C A / < %,,3+ > " / ? 7 # > " ? 7

*300 2C Schultz et al., 1999) mimicked the action of glibenclamide on

& & & & !,0 6 5 .- >K ; 31 < 31 ' N!"@ ' = "K 0 5 < < 5 ' 31 G#

motoneuron membrane potential, an effect accompanied by enhanced height of the antidromic spike (Figure 20b). On average, DPC significantly increased the membrane potential and resistance of motoneurons as indicated in Figure 20c (n=5). These data therefore demonstrated identical action of glibenclamide and DPC on spinal motoneurons.

1.5 Cl

-

dependent inhibition

$ < 8 . 9 E & " : A $- * < (00-+ A < " / 8 : 9 E

. $ ) "6 " 4,:0 * 1 ' ,#" ' ' ( 1 !# 2' ' * D ' I""#8G. " ; : _{< D < 8 ? <} < . * . ( $1+ " *? F < %,,-+ 7 . : . & ? (% . . . * " :(3Z1 =< [3+ 8 " * ? (% < + & ; : " 8 @ _{/ #}@ _{/ *7 " (003+}

8 " ? < D 8 . " . ) M / " " *E < (00-C # < (004+ < " M . : / " *E < (00-+ $ < 9 E . " :14Z% = * ['+< . " $ 1G' . *4Z( + :40 =< 3%Z(0 N< 9 E " :1Z% = ? (( . 8 : 9 E * :13 :40 = + * + (0 * + *? (% + " 9 E " * 9 E " ? (( + " < : _{9 E :} " / . : ; :

1.6 Activation of CFTR of interneurons

2 " : < / " 2 $ M * : +< . " ? 7 < < 2 ': : 2 *100 2C & < %,,,+ . 8 ? (- < / < . . . & :%0, * 5 0 . + $ " < ': : 2 .

. $ ) .+/+) "6 * '< A090 ?# ." ; 8 !" 8 1 < A090 '< ' 3 ' A090 ' ' G?8I""# . :%(4Z(3 * [4+ . 36Z%( N " / " ? 7< & ? 7 :%6( *3 2C 5 < (001C ? (- +< . : " ': : 2 *? (- + $ " < . " ': : 2 < . " / ? 7 %6( . 44Z6 * [3+ . *%(3Z%% N+C . " %0 .

( ( ( ( # 0#% $ 0#% ) 3 " )$ ) + , ' 8; ." ; H 0)1 ( ' ! * ? 5 ,>! ) !""! ' H 0)1 5 ,>!

1.7 CFTR in the neonatal rat spinal cord

8 7 8 * ? 7 8 C L < (00(+ . %60 : * % (+ 8 & ? 7 D *? (1 + D < 7 . . : * - 1 < " + " ? 7 7 " . * & . + 66Z- N " * [-+ 2 . ? 7 *? (1 < (:-+ . %'0 / (00 / < : ? 7 *2 < %,,1C A / < %,,3+ ? 7 .

> *? (1 < 1:3+ ) / . < . " . A -*? (1 < %+ ? 7 8 " . 1%Z3 N * [%%+ < . . 8 *%0Z( N+ . : 5 : ? 7 " " < *? (3+< . E) & ? 7 '0 N D . : ( * \ 0-3331+< * . (60 / C " ) < (003+ " : *2 & < (000+ " ? 7 " < " . . . . : *? (1 + : ? 7 ?- . D 8 & . < ? 7 : : . < ? 7 I / < " " . ? 7 8 ? D . * 8 < ) < . & + < < " < 8 ? 7 * < %,,6+ < / . < ? 7 8

, 0#% 1 (0 , ! ,>, (0 : K? (0 ' G * * ! K8(J KK ' , 1 ? # ' - 4 $ $ $ = 7 )= 0#%+$ ' ( J0 ) K! < ' 0 * ' ' J 0 '

2 Modeling results

. 8 " 8 8 < . / " ? 7 * # .+ ; :_{< 9 E * +} 9 E " " & *2 (00(+ 9 E : " < " D 8 * " . " F ) < (000+ " 8 " . " 8 . -" "

2.1 Soma

& " . 8 . < < -. & 2 " 5 8 -% 3%Z' (' %, 34Z3 3( < " * [6C < %,,'+ < . 5 G 8 D % 4,Z0 36< . % 11Z0 -1 # *%,,3+ % 6Z0 1 * // < %,,1+ " *%,,'+ .

#$ ( #$ ( #$ (

#$ ( 58 >

1-3 days 4-6 days 7-15 days references

16.9±1.9 m 19.6±2.0 m 23.3±2.5 m Fulton and Walton, 1986 21.7±3.0 m (n=89) - - Takahashi, 1990 17.9±0.6 m (n=60) 16.6±0.3 m (n=20) 19.2±3.5 m (n=16) Dekkers et al., 1994 a 22.7±3.64 m (n=250) - 26.0±4.22 m (n=285) Kerai et al., 1995 b, c 25.3±0.48 m (n=23) 24.1±5.37 m (n=45) 24.7±2.32 m (n=15) Safronov and Vogel, 1995 c - - 25.53±6.1 m _(n=7) Thurbon et al., ₁₉₉₈ d ' $ * ' ' ' C @s.d.= .s.e⋅ n G ' =

2.2 Axon

$. 8 . < )K ) / *%,,'+ 8 8 8 / * A+ . ' - 0 ' * + . %0 8 . 0 ' 300 D " . < " < # *%,6'+ # *%,6,+

8 < 8 : : A * // < %,,1C < %,,'+ - S 6 , S A< % %3 S ' 3 S A < . < . 8 8 D " " 8 . ? (4 < . A< 8

2.3 Proximal dendrites

. 8 ! < 8 8 8 // *%,,1+ *%,,'+ . " 8 *- %4Z% 1( 4 ,(Z% ',< " C Z C [%-+ // *%,,1+ " & . * %:,+ / 4 ,(Z% ', 8 8 " . . // *%,,1+< " * " . )K < %,,(+ * > .< %,,1+ > " : & " < *%,,'+ " *? (6 + " . * [0 '4< [6C X0 03C J / + A . " < ? (6 " " / .

? (6 . * C + - " 8 . " . 8 - _{* [0 '(< [6C X0 03+ < <} " *%,,'+ < . " < " Z $ < . " . < . ? (6 < * . +< . $ 8 " . < 8 D . 2 . . 8 - 8 * " . )K < %,,(+ " < . < & 8 * // < %,,1C > 9 < %,,(+ ? / < 8 . . < . D < ? (4 . 8 * / .+ 8

' ' ' ' # * ", ", ,"E * < * ' * C K3 0 * 0 B

3 3 3 3 + 8 $ 1 > @ ".H 3 !H! 3 O & < ' * 3 ,--H

2.4 Distal dendrites

8 . " < . . < . 5 < < ≤ 3 N *E . ? < %,'%C # F ]. < %,''+ . D " . 7 J

V(d_parent3/2 _,1+d3_parent/2 _,2)/d_parent3/2 W .

" *%,,'+ " 7 J D 0 ,(Z0 %1 0 ',Z0 %- // *%,,1+ " 8 < . % 6Z0 4 0 6Z0 % < " < 7 J < 8 . / . d_sibling/d_parent . 0 1 // *%,,1+ < < 8 < < d_parent⋅[0.4±rand(0.6)] * Z + 7 J *%,,1+ . " ? D . ! 8 8 < . % : // *%,,1+ *%,,'+ " " 8 D 3 %Z% - * [%-+ < . 5 " < " . * + 8 8 * " . )K F < %,,(+ > " : & *%,,'+ / . 8 E ? (6 * [0 '3< [6C X0 03+<

#$ #$ #$

#$ 0 8

parameter pooled experimental data model (n=25)

AD, µm2 6825±1918 * 8713±1990 AS, µm2 1788±893 1557±483 AD/AS 4.3±2.3 6±2 bmo 5.1±1.3 * _5±1 n-Prox 6.3±1.8 6.1±1.3 n-End 15.1±3.9 27.8±9 l-Tot, µm 2634±676 * _2447±842 M _{G,K < G>} ) ' L 03 80 8 * 8 1 * 8 D 8 * 8 + ? (' . 8 . & " $ ? (4 " " 1 ? (' . 8 " D

# $ % " 8

5 ' *

*

3

2.5 Motoneuron complexity defines synaptic input topography

< )K

*%,,(+ " 8 <

< . " . > 8 . J 7 " 8 8 8 8 " *) F %,,(+ // *%,,1+ & 8 . " 8 < ( ( ,< Q'0 S < 8 ((0:(10 S // J . - $ . / . / 8 8 8 8 . (3 S . " < . C 8 . 10 S C -0N 8 . 60 S . 8 '3 S (0N . . " " 8 A . " < " . 8 3 & 8 8 * ? (' + " 8 * - + (3 / " 8 . . '0:%00 S . // J

#$ #$ #$ -#$ - ! $ " ) INa, S/cm2 _IK A, S/cm2 IKdr, S/cm2 Soma 0.113 0.218 0.029 Proximal 0.003 0 0.001 Distal 0.003 0 0.001 AH 0.7 0 0.11 IS 0.7 0 0.11 Proper 0.012 0 0.04 3 < ' !""" 3 4 3 !""! 1 * ( :P Q : < ,--H ( * < 0J *

2.6 Examples of electrical activity generation

> 8 " D *E %,,6C < (00(C+< " : < < @_{< #}@ _{*# + : #}@ _{*# +} *)K F ) / < %,,'C " < (000+ 8 . ? (, * 4+ ;I7$ 8 " . . *%,,'+ . D * + . ( 1 S?G (_< . *7 + 3 - /Ω ( _{8 *7 + . '6}Ω . " / *:60 =+ ? (, . " 5 * .+< . G" 8 6' 2Ω . 8 * < %,,'+ < . " < " .

. " : . E *0 % + 8 * . ? (, + . " / *? (, + 0 3 3 *? (, + "

2.7 Modeling of CFTR inhibitors action on motoneurons input

resistance and spike overshoot

8 ? 7 ? 7 / " < . < . 8 " 2 . . ? (, < . 7 . *%44 Ω (_{+ .} (( 2Ω " < < ? < 7 *(%4 Ω (₊ . (-N * (6 2Ω+ ? 7 * 8 " . (,Z%0NC ? -0 + / " . " . *? -0 + & ? 7 . " / :61 =< . / *? -0 + D . 7 & * :61 =+< " / *? -0 + ? -0 . 5 0 %3 & :60 = / A . " < ( = " . "

" . 8 7 < . " (3N * . + ? < / 7 " 5 *? -0 + < " . " < . : * ? 7+ .

, " " 0 * (D7 $( 8 * !. !H ' = "", 0 8 ""# ""# 0 2; ' * J ", * '< < "# 0 # 5

(& (& (& (& " $ 2; ' < C 8 !-!! !>)Ω8 C < ' < < 8 ' < ' D < >" ; >? ; < < D < ' < ' = ' ' 1 *

* < '

Discussion

? 7 8 / ? 7 . / 8 ; :_{< 9 E * +} -8 . < " * " + < " 8 ? < & 8 9 E * + & &

Action of glibenclamide on the spontaneous activity in the

neonatal rat spinal cord can not be explained by K

+

conductance

block

< 8 . / " " : " *# + . " #@ _{8 * < %,''< (003C <} (004+ # < #@ _8< & < " * " < (001+ # " / . 8 " < : D *H < (00%C H F / < (00(+ $ * P < (00%+ *? < (001+ < " " . #@ _{> #}@ < " . <

< # " / " " # *A < (00%+ < " / " " . # * (003+ U # " < / # *E < (001+ 8 < 8 . 9 < " / 9 E & " " 8 : / " # & . . / 8 < < . " " < < . . *E < %,,4 < + " . / . @_G#@ _{*E < %,,6+} 2 8 . " # < " 8 : I & 8 #@ . / 8 . . " . #@ / 1: *1: + " / * F < (003+ 8 3:A *E < %,,4 +< 2 < *E < %,,4 + *E < %,,6+ " < . D " . / 8 .

8 #@ ₂

#

/ < :

. "

Changes in electrically or pharmacologically induced network

activity evoked by glibenclamide

. / 8 ; " . / # < & . . . . / *. / . + ? < . . #@ _" " < 9 E . / " . 8 #@ _/ A < * + 8 . 2 2 : . 9 E * +< 8 9 E : ? 7 . / # * & < %,,,+

CFTR as a Cl

-

regulator

* ? 7+< : _{< 8} < . < < < * F > <

%,,,C F < (00-+ . * F > < %,,,C F < (00-+ 9 ? 7 & : *) . < (00-+ ? 7 * & < %,,,+ " < ? 7 " " *A / < %,,3+< . # *2 " < %,,'C H F / < (00(+ < < ? 7 . . : _{9 E}

How could CFTR contribute to E

Cl-

in newborn spinal neurons?

: _{8 # (} " D *E < (00(C < (00-+ < : _{9 E < < .} " *E < (00(C 7 " < (003+ " < . < # ( 8 * + / : 9 E " / & *A < (00%C I < (00(C < (001+ E : _{9 E} *A < %,'-C > < %,,(C < (00-+< ; : _{" "} : _{. " " *:'6 =+<} & ; : $ . < 7 . . " " ; : _{. . 8 <} *9 F ] / : < %,,3+ ;" " ; : _{< "} G"

: : _/ " . / . / ? 7< . " $ . " . < ? 7 * & < %,,,+< < < . ? 7 :%6( * 5 < (001+ $ < . D ? 7 " ': : 2 " ? 7 * F > < %,,(C E < (000C < (001C > < (001+ . . & ? 7 :%6(< ? 7 8 . ? 7 : * F > < %,,,+ < ? 7 : . 8 < < < * F < (00-+< . 8 " ? 7 # % : _{" ;} : & 9 E * & < (003+ $ ? 7 # : " " * < (003+< . ? 7 8 # % * / F < %,,,+ & ! . " . < " ? < . 8 ? 7 " " " . & 9 E * + & < . ? 7 ; : ₈

CFTR transcripts and membrane expression in spinal tissue

? 7 *2 < %,,3C > < %,,,+ " < ? 7 7 8 " * < ? 7 :%6(+ ? 7 E ? 7 * < %,,0+ . 8 8 < : ? 7: " . . : " . " ? 7

Could the effects of glibenclamide be attributed to K

ATP

channel

block?

. # # 4 ( #@ _I7% : " * :E F E < %,,,+ # 4 ( 8 * & < (003+< 8 " / . " < # 4 (:G: _{. " *H <} (00%+ . # : 8 *H F / < (00(+ ? < . " " # * / < + D 8 / # " . . " # /

Functional implications

< 9 E & ; : _{" < 8} *2 < (00(+ ? 7 & ; : : 2 & " / 9 E * +: : " : " @ _* (@_{+ 9 E :} *7 < (00(+ E / . ? 7 . & 8 " " : : " @_* (@₊

Construction of neuronal model on the basis of available

morphological parameters

. : < " < 8 . . : : * " . # < (003+ : < . < / . *A < %,6,C E / < %,,(C < %,,,C < (00%+ > 8 < & A *%,6,+ . / . < * < : < < < . < . : +

* + " & " < < < " < " " " / . < " 5 *> F 9 < %,,(C // < %,,1C < %,,'+ E / J < . < " < " *E / %,,(+ " " / . *) < (003+ *)K F < %,,(+< " " . A J E / J " ) / . < J +$ ,-. * < (00%+ . . " < < 8 * 8 < 8 + . . 8 " " $ < 8 < . 8 < " E < " 8 I < " " 8 < "

Models of spinal motoneurons electrical activity

< . " *) ": " < %,'-C 7 F < %,''C E < %,,6C " < (000C < (00(+ " < " : & * " . " F ) < (000+< " 2 * + . < " . " " D *I < %,,1C ) / < %,,'C )K F ) / < %,,'+ " " " . $ " " : " = *%,,3+ " M: " @ _{. * : +} : * 7: + #@ _{" : "} " / *%,,0+ . * F < (004+ A . " < 8 " @ _{" 8} < : / D 8 * " < (000+ / 8 / 8 * )K F ) / < %,,'+ 2 *%,,3+ " / . < . @ . . 8 < / 2 /: )K F ) / *%,,'+ . @_: ^ 8 [ 600 G ( 8 /G

D " : < . / < @_{: ^ 8 [ %<(00} G ( _{D < "} @ 8 /G < . ^ 8. D 8 " *(000+ . & : : < "

Implication of electrical behavior modeling for explaining the

effects of CFTR inhibitors

" ? 7 . " " . < < 8 " . / ! . " . * 3 - /Ω ( _%44 Ω (₊ . 7 *%,',+ . 8 . " " 7 . %0:-0 7 " 8 * < %,'1+ . : D *I < %,,1C < %,,'C ) / < %,,'C )K F ) / < %,,'+ ? 7 . G 8 / 2 G 8 . . 8 " ? < ? 7 : _{& <}

< " " (, G ? *9 < %,,1+ D ? 7 * ? 7[% 1 G (+< " [( 1 S?G (_{* %,,'+<} : _{" :(0 = *;} :₊ D ? 7*=+_*; ::= +G < . = *:60 =+ ? 7 . *- =+ & 9 :A / :# & D " " . *#@_< @_< :_{+ * #< <} + / . ? 7 *%,''+ " D 0 %- 0 (3 G # G #< " I " :'6 = #@_{< @3- =} @ _{:(0 =} :_{*2 < (00% < "} < (000 < " +< 9 :A / :# & D :1% = < < -0 = " 8 < " L @_G#@ Q(3 = " *E < %,,6C + ? G # 0 %3 : 11 =< - = & ? 7 / : _/ > " . 8 / @ _{*2 < %,,3C} )K F ) / < %,,'+< / . / G 8 " G 8 / " < A . " < & * / " + 8 8 " G 8 & . " 5 < < . . 8 ? 7 8 . <

8 ? 7 2 8 : 8 ? 7 . < < *2 < %,,1< %,,3+ < 8 9 E 8 *E %,,4C " & %,,6+< 8 ? 7 . " / . :

References

9< $ . L< 7< # & # *(003+ : _{2 :} (@_{: 2%} / ! 160:16' :E )< E L *%,,,+ 2 : " - ! %0%:%-3 " & ?L< . ;< A < ? 7; *%,,6+ : & ( ! %30:%60 " &?L< . ;< 2 2 < ? 7;> *%,,,+ 7 . ! ! 6'6:6,6 9 *%,,,+ " / - ! %,3:(06 9 < # L)< L< ) *(00%+ 9 < 0 - 1 + 1 ! %%-%: %%13 ?2 *%,''+ 3P: : " / - ! ,6:%%' ?2 *(003+ : " ! ( # ! (016:(03' E < 7 I< > < U I *(000+ ? 7 : _{8 E !1--:110} E )< E ;< *%,,6+ / ! %6:(-E 2< *%,,,+ . " ! (0(,:(0-'

E < < . )L< ? & 2 *(00(+ & " " : ! %(1,:%(3' E : H *(00(+ ;8 9 E " ! - -! 6(':6-, E < = & / #< > < # $< 2 *(00-+ ( ! 1(,:110 E < > $< 2 A< ? L2 *%,,4+ ) & 9 E : ! !('-:(,6 E =< 7 & L< # $ *%,,6+ " (@_{: /} ! --6%:--'3 E ;< E )< *%,,4 + / ! 410:416 E ;< E )< *%,,4 + ) & . / ! 604-:6064 E ;< E 2< *%,,6+ " " ! -%36:-%46 E ;< E 2< *%,,'+ ;8 #@ _{: /} " ! . 2 3:A " ! (41-:(43( E . 9< ? 7; *%,'%+ " . : P ! %(%:%10 E L< = : > A< ] 9< E / < :E ) *(001+ . / . : " #@ _{* 2 ! %01:} %%(

E / 7;< 2 / > E< I / E *%,,(+ ! (10-: (1%4 E L< # $ *(00-+ ? : ! ,3-:,4-E L< ) L2< # $ *(00(+ $ & : . / - - !%06 %%6 MH< > . L7 *%,,1+ ! D " ( ! %1-:%36 A< 9 7L< 2 L< < & > < > 9 < $P7 7< ; *%,,0+ " ? 7 ( ! '(6:'-1 ? *(003 + 8 %! " " 3 4 2 !GG... G ^2 ^ B2 ^ [1(% ? *(003 + 8 (! (001 3 4 2 !GG... G ^2 ^ B2 ^ [1-, ?< ;< L?< = ) *(001+ " ! . ( ! -1-:-36 L < 7 L *%,',+ " < ! 4-:'6 =< 7 " < E : H *%,,-+ #@ _{& 8} 9 E ! 1,1:30-/ < A )< ; 2 *%,'6+ ( ! %0,:%%' < # L$ *%,6'+ 8 8 : : ! ('3:(,,

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