Recursos Necesarios

These ex p erim en ts were perform ed on a neurone in th e p ro th o ra c ic g an g lio n o f th e v e n tra l n erve cord of th e a d u lt male cockroach P erip la n eta americana. A s e c tio n of th e cord, co m prising th e th re e th o ra c ic g a n g lia and t h e i r c o n n e c tiv e s, was d is s e c te d o u t, p la c e d , v e n tra l s id e upperm ost, on a sm all p la s t ic s lid e , and a tta c h e d to i t by sm all ru b b er bands around th e s lid e and o v er th e c o n n e c tiv e s

(F ig u re 1).

To allo w e a s ie r a c c e s s to th e neurones under in v e s tig a tio n (which l i e tow ards th e p o s te r io r end o f th e v e n tra l s u rfa c e o f th e g a n g lio n ), th e p re p a ra tio n was a rra n g e d such th a t th e p ro th o ra c ic g a n g lio n was on a s l i g h t l y r a is e d p o rtio n of th e s lid e , w ith th e a n te r io r of th e co rd a t th e bottom o f th e b a th a s viewed through th e m icroscope. The p ro th o ra c ic g an g lio n was d esh eath ed to f a c i l i t a t e im palem ent o f th e c e l ls , and to a llo w a p p lie d a g e n ts a c c e ss to th e s u rfa c e membrane. I f th e s h e a th were n o t removed, i t s ro le a s a b lo o d /b ra in b a r r i e r would r e s t r i c t a c c e ss of a t l e a s t some of th e ex p erim en tal , a g e n ts

(T reherne & P ichon, 1972).

In o rd e r to a llo w f r e e r a c c e ss fo r dru g s and o th e r a g e n ts c ir c u la t in g in th e s a lin e , th e c e l l body of th e neurone was cle a n e d , and g l i a l tis s u e removed from around i t , e s p e c ia lly below th e soma. T his was

c

\

plastic

slide

^preparation

elastic

band

Figure The ventral nerve cord mounted on the plastic slide, The ganglion is 1,5-2 mm in diameter, and the slide is approximately 1 cm wide and 2 cm long,

ach iev ed u sin g a s a l i n e - f i l l e d m ic ro e le c tro d e , th e t i p o f which was broken back to a llo w e je c tio n , under p re ssu re from a sy rin g e , o f a d ir e c ta b le j e t o f s a lin e .

In some ex p erim en ts, a broken and f ir e - p o lis h e d m ic ro e le c tro d e , connected by tu b in g to a m outhpiece, was used to p ro v id e a vacuum to r a is e th e c e l l body above th e g e n e ra l le v e l of th e g an g lio n . T his improved a c c e s s f o r a g e n ts which were c a r r ie d in th e b a th c i r c u la tio n , b u t th e te c h n iq u e proved d i f f i c u l t to s ta n d a rd is e , and i t was l a t t e r l y abandoned in fav o u r o f com prehensive c le a n in g w ith a s a lin e j e t .

2 .2 THE EXPERIMBHTAL BATH

For th e se ex p erim en ts, th e p re p a ra tio n was mounted in a P erspex b ath , w ith a volume of 3 ml. P ro v isio n was made fo r c ir c u la tio n , oxygenation and rep lacem en t of th e s a lin e ( d e ta ile d co m p o sitio n s of a l l s a lin e s o lu tio n s used a re g iv e n in Appendix 1 ), as w ell a s d ir e c t in je c tio n of drug s o lu tio n s (F ig u re 2 ), The p re p a ra tio n w ith in th e b ath was viewed th ro u g h an Olympus zoom b in o c u la r d is s e c tin g m icroscope, f i t t e d w ith x20 ey ep ieces, g iv in g a range o f m a g n ific a tio n o f 14 to 80 tim e s. T his allow ed a c c u ra te p o s itio n in g under v is u a l c o n tro l o f m ic ro e le c tro d e s and drug p ip e tte s w ith re s p e c t to th e p re p a ra tio n .

The p re p a ra tio n chamber was p h y s ic a lly is o la te d fro m .th a t c o n ta in in g th e b ath e le c tr o d e s , and th e two were connected e l e c t r i c a l l y u sin g a g la s s b rid g e f i l l e d w ith an agar/K C l g e l. T his p re v e n te d th e g e n e ra tio n of v a ria b le J u n c tio n p o te n tia ls by a l t e r a t i o n s in th e io n ic environm ent of th e b a th e le c tr o d e s d u rin g exp erim en ts.

¥ suction^ =

Ogin

i

Ü .saline in plastic side i

Figure 2 The Perspex experimental chamber, Provision is made for the addition of agents to the circulation, and for the continuous superfusion and oxygenation of the circulating solution,

2 .3 ÎSTRACELLÜLâJR RECORDING

I n tr a c e ll u la r re c o rd in g s were made from th e neurone soma by s in g le o r dual im palem ent. G lass m ic ro e le c tro d e s were p u lle d u sin g a N arish ig e PE2 v e r ti c a l p u lle r from 1 ,5 mm e x te rn a l d ia m ete r th in -w a lle d fila m e n t g la s s c a p i lla r y tu b e , su p p lie d by C lark E lectro m ed ica l In stru m en ts. R ecording m ic ro e le c tro d e s, f i l l e d w ith 1 o r 2 M potassium a c e ta te , had t i p r e s is ta n c e s between 20 and 30 MQ.

Potassium a c e ta te was chosen a s th e f i l l i n g s o lu tio n s in c e i t has been noted th a t some in s e c t neurones show ex cep tio n al- s e n s i t i v i t y to changes in in te r n a l c h lo rid e le v e ls (Pitm an, p e rso n a l o b s e rv a tio n ). In a d d itio n , t h i s s o lu tio n was le s s lik e ly to a f f e c t th e r e s tin g p ro p e rtie s of th e c e l l , a s p o tassiu m c o n c e n tra tio n s in s id e a re h igh, and a c e ta te i s not r e a d ily tra n s p o rte d by p h y s io lo g ic a l mechanisms and cannot tr a v e l th ro u g h c h lo rid e ch an n els,

2 .3 .1 B e v e llin g H ic ro e le c tra d e s

In experim ents u sin g two m ic ro e le c tro d e s, w hether under c u r r e n t- o r vo ltag e-clam p c o n d itio n s , th e c u rre n t-p a s s in g m ic ro e le c tro d e was fre q u e n tly b e v e lle d to betw een 8 and 15 Mft, u sin g a slo w ly re v o lv in g (a 1 r e v /s ) f in e a b ra s iv e d is c . T his was viewed th ro u g h a b in o c u la r m icroscope under an g led illu m in a tio n from a lamp so p o s itio n e d a s to c a s t a shadow of th e m ic ro e le c tro d e t i p onto th e s u rfa c e of th e d is c , th u s allo w in g th e m ic ro e le c tro d e to be low ered u n ti l i t s t i p was j u s t in c o n ta c t w ith th e a b ra s iv e ,

2 .3 .2 H lc ro e le c tro d e H o ld ers

The m ic ro e le c tro d e s were lo c a te d in h o ld e rs made from f le x i b le s ilic o n e tu b in g , th ro u g h th e c e n tre of which a s i l v e r w ire was passed. T his in tu r n was d i r e c t l y so ld e re d onto a 4 mm p lu g , to allow fo r co n n ec tio n to th e probe s ta g e of th e re c o rd in g system . To e lim in a te ju n c tio n p o t e n ti a l s and b a s e -lin e d r i f t a s s o c ia te d w ith p o la r is a tio n , th e s i l v e r w ire was co ated w ith s i l v e r c h lo rid e by e l e c tr o l y s i s in a b a th o f 1 H p o ta ssiu m c h lo rid e ,

2 .3 .3 A m p lific a tio n

The probe s ta g e s of- th e a m p lifie rs were h eld in P r io r m icro m an ip u lato rs f i t t e d w ith 10 to 1 re d u c tio n d riv e s to g iv e f in e r c o n tro l in o rth o g o n al h o r iz o n ta l p la n es.

Each m ic ro e le c tro d e was co n n ected to a la b o r a to r y - b u ilt h ig h impedance, u n ita ry g a in a m p lif ie r . A b rid g e c i r c u i t was in c o rp o ra te d in to t h i s a m p lifie r to e n a b le th e membrane p o te n tia l of th e c e l l to be m onitored, and a t th e same tim e to allo w p o la r is a tio n of th e c e l l membrane by c u rre n t in je c tio n . The a m p lifie rs had p ro v is io n f o r c a p a c ita n c e com pensation, m ic ro e le c tro d e r e s is ta n c e check, and c a lib r a tio n (p re s e t 10 and 50 mV o u tp u t to CRO o r c h a rt re c o rd e r). A com plete c i r c u i t diagram i s g iv e n in Appendix 4.

2 .3 .4 G en eratio n o f C u rre n t P u lse s

Square-wave p u ls e s , of v a r ia b le am plitude and d u ra tio n were p ro v id ed by a G rass SD9 s tim u la to r, th e o u tp u t o f which was n o t is o la te d . For use in im paling th e c e l l , a p u lse o f h y p e rp o la ris in g c u rre n t, of 25 nA am plitude and 20 mS d u ra tio n , was used to d is r u p t th e membrane s u f f i c ie n t ly to allo w th e m ic ro e le c tro d e t i p to p e n e tra te .

Once an im palem ent had ta k e n p la c e , a m easure o f th e input, r e s is ta n c e of th e c e l l co u ld be o b ta in e d u sin g p u ls e s , a t a freq u en cy of 1 Hz, a t an am plitude s u f f i c i e n t to produce 10 mV h y p e rp o la ris in g ex c u rsio n s of membrane p o te n tia l a t th e s t a r t o f th e experim ent,

2 .3 .5 C urrent-C lam p R ecording

2 .3 .5 .1 S in g le M ic ro e le c tro d e R ecording

In p re lim in a ry in v e s tig a tio n s in to th e n a tu re of th e resp o n se to dopamine, s in g le -m ic ro e le c tro d e c u rre n t-c la m p re c o rd in g s were made. A s in g le m ic ro e le c tro d e , f i l l e d w ith 1 o r 2 M p o ta ssiu m a c e ta te was used, connected a s d e ta ile d above. T his gave a measure o f th e r e s tin g membrane p o te n tia l of th e c e l l and any e f f e c t s o f dopam inergic a g e n ts on i t . I t was a ls o p o s s ib le to ap p ly re p e a te d