Figure 9 Representative data indicating the current-voltage relationship of the cell under voltage-clamp in the presence and absence of dopamine^ and the voitage-dependence of the resultant dopamine-induced current, represented as a difjfrence curve. Data were obtained by the 'short- pulse' method,
Current (nA)
30 T 10- - -30 -100 0 60 -150Command Potential (mV)
Figure 10 A difference curve indicating the voitage-dependence of the voltage- clamp response current to pressure-applied dopamine, obtained by the
'short-pulse' protocol,
c u rren t resp o n se r e s u ltin g from th e mic ro ion to p h o re sis was n o t m a te ria lly d if f e r en t from th a t o b tain e d by p re s s u re a p p lic a tion , and th e vo ltag e-d ep en d en cy of th e resp on se was a ls o sim ila r . F ig u re 11 shows a ty p ic a l v o ltag e-clam p resp on ses,
3 .1 .4 C o n tro l E xperim ents
To en su re th a t th e unusual shape of th e IV curve f o r dopamine was n o t a r te f a c tu a l, th re e dif f e r en t f a c to r s were I n v e s tig a te d . -These were th e p resen ce of th e s t a bi l i s e r (1% sodium m e ta b is u lp h ite ) in th e drug s o lu tion , th e pH of th e s o lu tion , and th e p hy s ic a l e f f e c t of th e p re s s u re -p u ls e on th e c e l l membrane,
3 ,1 ,4 ,1 C o n tro l fo r S t a bi l i s e r
In o rd e r to e lim in a te th e s t a bi l i s e r a s a cau se o f a r t e f a c ts , ex p erim en ts were perform ed u sin g a s o lu tion of dopamine in s a lin e , without a s t a b i l i s e r . These experim ents were o f n e c e s sity ra p id , s in c e th e dopamine s o lu tion became tin g e d with p in k v ery soon a f t e r
i t had been made up, in dic a tin g th a t o xid a tiv e deco m p o sition o f th e dopamine had begun. A fte r 20 to 30 minutes, th e s o lu tion no lo n g e r e li c i t e d any resp on se even when a p p lie d in lo n g p u ls e s , s u g g e stin g th a t most of th e dopamine p re s e n t had decomposed.
In term s of th e siz e and tim e co u rse of in div id u a l re sp on se s, and of th e voltag e-d ep en d en cy of resp on ses over a ran g e o f command p o te n tia ls , th e r e s u l t s o f th e s e ex p erim ents, under b o th c u r r e n t- and
A.
B.
C.
11
nA10s
MP ”60
mVFigure 11 Voltage-clamp responses to the microiontophoresis of dopamine, Dopamine was applied using an ejection current of 250 nA (trace A) or 100 nA (traces 8 & C), and a retaining current of 20 nA, Pulse duration was 1 s, and holding potential -60 mV. It proved impossible to eradicate completely the large current artefact resulting from the ejection current pulse, due to the interference this produced with the clamp current,
voltag e-clam p , were in dis tin g uis h a b le from th o se o b ta in e d u sin g a dopamine s o lu tion c o n tainin g th e s t a bi l i s e r (F ig u re 12).
E xperim ents were a ls o perform ed in which th e s t a b i l i s e r s o lu tion alo n e (1% sodium m e ta b is u lp h ite in s a lin e ) was p re s s u re a p p lie d onto th e s u rfa c e of th e neurone soma, w hile re c o rd in g s were made under cu rre n t-c la m p o r v o ltag e-clam p c o n dition s . These ex p erim en ts produced no evid en ce o f any resp on se, ei t h e r d e p o la ris in g o r hy p e rp o la risin g , o r o f any e f f e c t on th e in p u t r e sis tan c e of th e c e l l.
3 ,1 .4 .2 C ontrol f o r pH
G reat c a re was ta k e n (with in ex p erim en tal lim ita tion s ) to c o r r e c t th e pH of a l l drug s o lu tion s . T his was done in th e p re p a ra tion of th e sto c k s o lu tion s , such a s th e s t a bi l i s e r , e i t h e r by th e use o f a c a r e f u lly c a lib r a te d pH m eter, o r by th e use o f pH pap er and sta n d a rd b u ffe r s o lu tion s . In th e case o f v ery sm all am ounts o f s o lu tion , pH was measured by pH p ap er, s in c e a micro-pH m eter was not a v aila b le . The s o lu tion was c o rre c te d to a pH a s n e a r to 7 .2 a s p o s sib le (equal to th a t o f norm al p hy s io lo g ic a l s a lin e ) .
To e lim in a te th e rem ote p o s si b i l i t y th a t a d r i f t in th e pH o f drug s o lu tion s co u ld have been re s p o n sib le f o r th e re sp on se s o b tain e d , some ex p erim en ts were a ls o perform ed u sin g a s o lu tion o f s a lin e , with added HCl o r NaOH, to a l t e r th e pH to e i t h e r 6 .7 (0 .5 units le s s th an norm al), o r 7 .7 (0 .5 u nits more th an norm al). A ty p ic a l c u r r en t-
10 mV
20s
Figure 12 A representative trace of a current-clamp response produced by pressure-application of a solution containing dopamine without stabiliser, Current pulses were applied through a second microelectrode, and resting potetial was -65 mV,
clamp experim ent i l l u s t r a t e s th a t no d e te c ta b le e f f e c t was seen (Figure 13),
3 ,1 ,4 ,3 C on tro l f o r P re ssu re A rte fa c ts
In o rd e r to t e s t fo r th e e f f e c t s of p re ssu re on th e neurone, s a lin e was eje c te d onto th e s u rfa c e o f th e c e l l.
Under both v o lta g e - and c u rre n t-c la m p c o n dition s , a d e f le c tion in th e r e s tin g s t a t e o f th e c e l l membrane was seen o n ly when hig h e je c tion p re s s u re s were used ( > 15 p si) , o r when th e eje c tion pip e tte was very c lo se to th e c e l l s u rfa c e . As in dic a te d in Fig u re 14, th e change in membrane p o te n tia l caused by a p re s s u re e je c tion was e s s e n ti a lly of th e same d u ra tion a s th e p re s s u re p u lse . I t showed none of th e v o ltag e-d ep en d en cy a s s o c ia te d w ith th e dopamine resp o n se. Since th e normal tim e co u rse of a s in g le dopamine resp o n se was o f th e o rd e r o f 30 seconds, and p re s s u re p u ls e s were r a r e ly lon g e r th a n 1 second, i t was u s u a lly p o s s ib le to e lim in a te th e s e a r t e f a c t s in normal ex p erim en ts. In c a s e s where i t d id prove im p o ssib le , th e p re p a ra tion was r eje c te d .
The ex p erim en ts in v o lvin g th e mic ro ion to p h o re sis and b a th a p p lic a tion o f dopamine a ls o serv ed a s c o n tro ls fo r t hi s , s in c e re sp o n se s were o b tain e d in b o th c a se s which c le a r ly co u ld n o t be a t t ri b u t e d to th e e f f e c ts o f p re ssu re .
pH 6 7
I # # #
pH 7 2
pH 7 7
Figure 13 An illustration of the lack of membrane electrical response to solutions of saline at different pH values, Current pulses were applied through a second microelectrode, and there was no evidence of any alteration in membrane resistance. Resting potential was -62 mV,