TT
5mV
10 s
Figure 14 A typical current-clamp pressure artefact, The transient change in membrane potential (resting potential -59 mV) Is of the same duration as the pressure pulse.
3 , 1 , 5 Ion S u b s t it u t io n E x p erim en ts
The ex p erim en ts id e ta ile d h ere took th e form of ion - s u b s titu tion s in which e x te rn a l c o n c e n tra tion s of S'a’*', K*^, Cl~ o r Ca^^ were a lte r e d .
In a d dition , in t e r n a l c o n c e n tra tion s of Cl" and Ca^^ were changed by im palement with m ic ro e le c tro d e s c on tainin g KCl and tri- p o ta s s iu m c i t r a t e re s p e c tiv e ly . The calciu m an ta g o nis ts cadmium and verapamil were a p p lie d , to d is c o v e r th e e x ten t to which Ca^^ was in v o lv ed ei th e r in th e ini t i a t i on o f th e response or in th e c a rry in g of c u rre n t d u rin g i t . These two a g en ts were chosen sin c e i t has been shown th a t th e re a re dif f e r en c e s in th e ways in which o rg anic and
in o rg a nic calciu m channel b lo c k e rs a c t (Oyama e t a l , 1986). The c on s titu tion s o f th e rep lacem en t s a lin e s used a re given in Appendix
1.
F in a lly , ex p erim en ts were perform ed using a co m p u ter-d riv en p u lse g e n e ra to r with f a s t tim e r e s o lu tion , which en ab led th e e f f e c t s of dopamine on o v e r a ll membrane c u r r e n ts to be a sse sse d .
3 .1 .5 .1 E ffe c t of A lte ra tion o f [E a^]«
E xperim ents were perform ed in which th e p re p a ra tion was in c u b ated in a s o lu tion in which [N a^]o was reduced from a normal v alu e of 214 mK, to 42.8 mM, a fi v e - f o l d re d u c tion . In th re e ex p erim en ts, a f t e r
in c u b a tion in 42.8 mM ha"^ s a lin e fo r up to 15 minutes, th e inw ard c u rre n t component seen a t command p o te n tia ls in th e re gion of -20 mV was su p p resse d , w hile in fo u r o th e rs i t was un a ffe c te d . Fig u re 15
40 30 20 10 0 -10 -20 “30
Current (nA)
Legond —H Control 6 mine 42.8mM Na 10 tnlns waeh '» » » f ' »'■ -40 60 -200 -150 -100 -60 0Command Potential (mV)
Figure 15 The effect of a five-fold reduction in CNa-^lo, from 214 mM to 42,8 mM, on the dopamine-induced response current at a range of command potentials (holding potential was -60 mV), Data were obtained by the ‘short-pulse’ protocol, This illustrates an experiment in which the inward current at positive potentials was altered by the ion replacement, In other experiments, no effect was seen,
i l l u s t r a t e s an experim ent in which su p p re ssion o ccu rred in low sodium s o lu tion . No e f f e c t was observed upon th e in c re a s e in m agnitude of th e c u r r en ts a t more n e g a tiv e command p o te n tia ls ,
3 .1 .5 ,2 E ffe c t of A lte ra tion o f [K^lo
E xperim ents were perform ed in which [K*^‘3o was in c re a se d from a normal v alue of 3 .1 mM to 15.5 mM, In fiv e experim ents t hi s s u b s t itu tion caused th e re d u c tion o r a b o lition of th e inw ard c u rre n t kin k , and one of th e se i s i l l u s t r a t e d in Fig u re 16. However, in th r e e o th e r exp erim en ts t hi s re d u c tion was n o t observed. Like low Ma'^ s o lu tion s ,
i t d id n o t, however, ap p ear to have any e f f e c t on th e m agnitude o f th e inw ard c u rre n t a t more n e g a tiv e p o ten tia ls .
3. 1 .5 .3 E ffe c t o f A lte ra tion o f CC1~3<=. and CCl~]t
In th e se ex p erim en ts, CCl“ 3c was reduced from a normal le v e l o f 235 mM to a le v e l of 1/5 norm al, 47 mM. Micro electro d e s f i l l e d w ith 3 M KCl were used in o rd e r to r ais e CCl“ l i , alth o u g h i t was n o t p o s sib le to e s tim a te by how much CCl~]j. was being a lte r e d . The r e s u l t s of both s e t s of e x p erim en ts were th e same - in some c a se s, (3 out of 7 fo r changes in CCl“ 3o and 3 o ut of 8 fo r changes in tC l~ 3i> , th e p o in t a t which th e m agnitude o f dopamine induced c u r r e n ts in c re a se d d ra m a tic a lly was s h if te d in th e d e p o la ris in g dir e c tion . Fig u re 17 shows a ty p ic a l re c o rd in which CCl"3o was a lte r e d . In th re e f u r th e r ex p erim en ts, no e f f e c t was observed. Mo e f f e c t was seen on th e m agnitude of c u r r en ts a t p o te n tia ls p o s itiv e to th e r e s tin g p o t e n ti a l .
3 0 - r
Current (nA)
20 10 Legend ‘H ~ Control 10 mlna 16.6mM K* • 10 mine wash $ $ a * ■h 80 I ( -H -l • ( I 4— 1 1 I l- l ' l I 1 ■■■( I I I I I -160 -134 -118 -lo a “8? -71 -66 -30 -24 -8 +H -H8 23Command Potential (mV)
Figure Î6 The effect of a five-fold increase in CK*]*, from 3.1 to 15,5 mM, on the dopamine-induced response current at a range of command potentials (holding potential was -60 mV), In this experiment, this change reduced the inward current at positive potentials, In other experiments, however, no effect was observed.