CAPÍTULO I Planteamiento Teórico
4.1.1.2 Comunicación Interna Descendente
-20 - Control 10 rnins Butaclamol 15 mins wash -30 — -40 -200 -150 -100 -50 0 50
Com m and Potential (m V)
Figure 54 The suppressive effect of 1 x 10“^ M butaclamol on the voltage-clamp response to dopamine at at range of command potentials, Holding potential -60 mV,
c on tro l c u rre n t m agnitudes o f -30 nA a t a command p o te n tia l of -140 mV, -0 .5 nA a t -60 mV, and +10 nA a t +30 mV, were reduced to 0 nA a c ro s s th e f u l l range of membrane command p o te n tia ls a f t e r 10 m inutes exposure to 1 x 10"^ M b u taclam o l. Following 15 minutes wash, th e resp o n se was r e s to r e d to v a lu e s of -28 nA a t -140 raV, -0 .5 nA a t -60 mV, and +8 nA a t +30 mV, Bfo e f f e c t was observed on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip of th e c e l l.
F lu p en thix o l was found to be a c tiv e , under both c u r r e n t- and v o lta g e - clamp c o n dition s (6 ex p erim en ts each under v o lta g e - and c u r r e n t- clamp, and 4 a d dition a l t e s t s fo r each of th e low er c o n c e n tra tion s down to 1 X 10“ '^), a t c o n c e n tra tion s a s dil u te a s 1 x 10“ -’ M. A fte r in c u b a tion fo r 20 minutes, a t a c o n c e n tra tion o f 1 x 10“^- M, i t re v e r s ib ly su p p re sse d th e resp on se under c u rre n t-c la m p (Fig u re 5 5 ). In a ty p ic a l v o lta g e -c la m p experim ent (Figure 5 6 ), b e fo re exposure to 1 X 10“® M flu p en thix o l, th e c u rren t m agnitudes were -35 nA a t a command p o te n tia l of -150 mV, -0 ,5 nA a t -60 mV, and +11 nA a t +30 mV, Following 10 minutes exposure to 1 x 10“ ® M flu p e n thix o l, th e se v a lu e s were red u ced to 0 nA a t a l l command p o te n tia ls . A fte r 15 m inutes wash, th e resp o n se c u rre n t m agnitudes re tu rn e d to norm al, with v a lu e s of -33 nA a t -150 raV, -0 .5 nA a t -60 mV, and +10 nA a t +30 mV. No e f f e c t was seen on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip of th e c e l l .
3 .3 .2 .4 An ta g o nists A ctive a t Di R eceptors
As in dic a te d in S e c tion 3 .2 .3 ,1 , SCH 23390 proved to be a p o te n t and s e le c tiv e dopam inergic an ta g o nis t. Under c u rre n t-c la m p c o n dition s ,
:DA 10mV DA
15s
DA
Normal Flupenthixol IpM
20 m in.
lOmin.wash
Figure 55 The suppressive effect of 1 x 10"® M flupenthixol on the current-clamp response to dopamine, Current pulses were applied through a secon d microelectrode, and resting potential was -57 mV,
Current (nA)
20 T 10 - - -10 H — Control 10 mins Flupenthixol 15 mins wash -30 — -200 -150 -100 0 50Com m and Potential (m V)
Figure 56 The suppressive effect of 1 x 10"® M flupenthixol on the voltage-clamp response to dopamine at range of command potentials, Holding potential -60 mV,
a t a c on c e n trâ t ion o f 1 x 10~® H, SCH 23390 r e v e r sib ly suppressed th e resp on se to dopamine, and washed o ff co m p letely a f t e r 10 to 20
m inutes (Fig u re 3 4 ). At t h i s c on c e n tra tion , th e resp o n se was r e li a b l y and re p e a ta b ly a b o lish e d . At c on c e n tra tion s p ro g re s siv e ly more d il u t e th an t hi s , th e e f f e c t dim inished, un ti l a t 1 x 10~® M, i t was v i r t u a l l y un d e te c ta b le .
Under v o lta g e -c la m p c o n dition s , th e normal IV curve due to dopamine was f la tte n e d o u t co m p lete ly a t a c on c e n tra tion o f 1 x 10"^ M,
in dic a tin g a t o t a l blockade o f th e resp o n se even a t r e l a ti v e l y
n e g a tiv e command p o te n tia l s , where th e resp on se c u r r e n t would be ex p ected to be a t i t s la r g e s t , In a ty p ic a l experim ent (Fig u re 3 5 ), b e fo re th e a p p lic a tion of SCH 23390, a t a command p o te n tia l o f -160 mV, th e m agnitude o f th e induced c u rre n t was -13 nA, - 0 .5 nA a t -60 mV, and +15 nA a t +30 mV. A fte r 5 minutes exposure to 1 x 10“® M SCH 23390, th e s e v a lu e s were red u ced to 0 nA a t a l l p o te n tia ls . A fte r 10 m inutes wash, th e resp o n se had been re s to re d , giv in g resp o n se c u rre n t m agnitudes of -1 0 nA a t -160 mV, - 0 .5 nA a t -60 mV, and +10 nA a t +30 mV, No e f f e c t was seen on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip o f th e c e l l .
SCH 23390 was te s t e d , in one experim ent only, on th e resp o n se o f neurone D3 to apom orphine, under v o ltag e-clam p . In t hi s ex p erim en t, a c o n c e n tra tion o f 1 x 10“® M SCH 23390, a f t e r in c u b a tion f o r 7 min u tes, signi f i c an t ly reduced th e resp on se c u r r e n ts under v o lta g e -c la m p o v er th e com plete s ta n d a rd range o f command p o te n tia ls . A lthough t h i s r e s u l t i s un s u b s ta n tia te d s in c e i t i s from a s in g le
experim ent, i t i s in dic a tiv e t h a t th e re c e p to r upon which apomorphine
i s having i t s e f f e c t i s b ein g b locked by SCH 23390.
3 .3 .2 .5 An ta g o nists A ctive a t Da R eceptors
H alo p erid o l su p p re sse d th e re sp on se of neurone D3 t o dopamine under bo th c u r r e n t- and v o lta g e -c la m p c o n dition s <6 ex p erim en ts e a c h ), The e f f e c t was observed a t c o n c e n tra tion s a s low a s 1 x 10“® M. In a ty p ic a l v o ltag e-clam p ex p erim en t (Figure 5 7 ), th e c on tro l v a lu e s of Induced c u r r e n t were -26 nA a t a command p o te n tia l o f -160 mV, -0 .5
nA a t -60 mV, and +15 nA a t +30 mV. F ollowing 15 minutes exposure to 1 X 10“® M h a lo p e rid o l, th e resp on se c u rre n t was reduced to 0 nA a t a l l command p o te n tia ls . A fte r 20 m inutes wash, th e resp o n se was re s to re d , giv in g v a lu e s o f -1 7 nA a t -160 mV, - 0 ,5 nA a t -60 mV, and +10 nA a t + 30 mV, Under c u rre n t-c la m p , th e m agnitude of th e d e p o la r is a tion was co m p lete ly su p p re sse d by exposure to 1 x 10~^ M h a lo p e rid o l, and re c o v e re d co m p lete ly on w ash-out (Fig u re 58). No e f f e c t was seen on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip o f th e c e l l .
Under b o th c u rre n t-c la m p and v o ltag e-clam p c o n dition s , s piro p e rid o l produced an ir r e v e r s i b le b lockade of th e resp on se (7 exp erim en ts eac h ). In a r e p r e s e n ta tiv e v o ltag e-clam p experim ent (Fig u re 59), th e c on tro l ru n produced v a lu e s f o r th e induced c u rre n t m agnitudes o f -25
nA a t a command p o te n tia l o f -150 mV, -0 .5 nA a t -60 mV, and +14 nA a t +30 mV, A fte r 15 minutes exposure to 1 x 10“ ® M s pir o p e r id o l, th e resp on se c u rre n t m agnitudes were reduced to 0 nA a t a l l command p o te n tia ls . Follow ing 45 min u tes wash, th e resp o n se was s ti l l 0 nA,
20
Current (nA)
10 - - —t— Control - 0 - 5 mins Haloperidol 45 mins wash -80 -200 0— 0 —0 — -150 -100 -50 50Com m and Potential (m V)
Figure 57 The suppressive effect,of \ x 10"^ M haloperidol on the voltage-clamp response to dopamine at range of command potentials, Holding potential -60 mV,
Control
15min 10 ^ 10min
wash'
HAL
Figure 58 The suppressive effect of I x HQr* M haloperidol on the current-clamp response to dopamine, Current pulses were applied through a second microelectrode, and resting potential was -56 mV,
2 0 -I 10
Current (nA)
-10 - -30 -200 -150 -100 -50 0Com m and Potential (m V)
Legend _ Control
- 0 - 15 mins Spiroperidol 45 mins. wash
A A A y:(. .A_A_A....A.. A__XX__A X5? ...,.^.. _0-7<]
■} I l> 0 0 ' 7 1 • 1 f 4- I : 50
Figure 69 The suppressive effect of 1 x 10"® M spiroperidol on the voltage-clamp response to dopamine at range of command potentialsi The effect was not reversible, Holding potential -60 mV,
and showed no signi f i c an t sign of reco v ery . T h is e f f e c t was re p e a te d under c u rre n t-c la m p c o n dition s . No e f f e c t was seen on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip o f th e c e l l ,
H etoclopraraide, a t c on c e n tra tion s in ex ce ss of 10"^ M, and
in c u b a tion tim e s in e x c e ss o f 40 minutes, had no d e te c ta b le e f f e c t on eith e r th e d e p o la ris in g resp o n se to dopamine under c u rre n t-c la m p , o r th e dopamine-induced c u r r e n ts seen under v o ltag e-clam p a c ro s s th e f u l l range o f membrane command p o te n tia ls <9 ex p erim en ts eac h ). A ty p ic a l v o lta g e -c la m p ex p erim ent (Figure 4 1 ), shows th a t th e m agnitude o f th e in duced c u r r e n ts was n ot a f f e c te d by exposure f o r up to 25 m inutes to 1 x 10"^ M m etoclopramide, lîo e f f e c t was seen on th e r e s tin g c u rre n t- v o lta g e r e la tion s hip of th e c e l l.
Under b o th c u r r e n t- and v o ltag e-clam p c o n dition s YH 09151-2 r e v e r sib ly su p p re sse d th e re sp on se to dopamine (8 exp erim en ts e a c h ). Under c u rre n t-c la m p , th e siz e and d u ra tion of th e resp o n se was reduced (Fig u re 6 0 ), In a re p r e s e n ta tiv e v o ltag e-clam p experim ent (Figure 6 1 ), th e m agnitude o f th e dopam ine-induced c u r r e n ts was su p p ressed over th e com plete range of command p o te n tia ls . P r io r to th e exposure of th e p re p a ra tion to YM 09151-2, th e c u rre n t m agnitudes were -1 2 .5 nA a t a command p o te n tia l of -160 mV, -0 .5 nA a t -60 mV, and +15 nA a t +30 mV. F ollowing 5 minutes exposure to 1 x 10~® M YM 09151-2, th e v a lu e s were red u ced to 0 nA a t a l l command p o te n tia ls , A fte r 10 m inutes wash, th e resp o n se re tu rn e d , and th e v a lu e s were -10 nA a t -160 mV, - 0 .5 nA a t -60 m¥, and +10 nA a t +30 mV. No e f f e c t was seen on th e r e s tin g c u rre n t-v o lta g e r e la tion s hip o f th e c e l l.