Clasificación de la microflora edáfica

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“200 “too "50 0 6 0

Command Potential (mV)

Figure 29 The voltage-dependence of the voltage-clamp response to octopamine. The curve was obtained from the difference between the magnitudes of current obtained by voltage pulses in the presence and absence of dopamine, Holding potential was -60 mV,

p o ten tia l of th e c e l l , th e a p p lic a tion of octopamine induced a sm all

inw ard c u rre n t. In g e n e ra l, th e m agnitude of t h i s was s m a lle r fo r a given a p p lic a tion siz e th an was th e c u rre n t induced by dopamine, commonly around 0.25 nA compared to 0.5 nA fo r an e q uiv a len t a p p lic a tion o f dopamine. At command p o te n tia ls p ro g re s s iv e ly more

n e g ativ e th an th e r e s tin g p o te n tia l, th e c u rren t rem ained a t around 0.25 nA. A p oin t was reach ed , however, u s u a lly between -130 mV and - 160 mV, a t which th e m agnitude of th e induced inw ard c u rre n t

in c re a se d su d d en ly , in a sim ila r fa sh ion to th a t observed with th e a p p lic a tion o f dopamine. In th e case of octopamine, however, th e m agnitudes of th e induced c u r r e n ts a t given p o te n tia ls were s m a lle r, th e r a te a t which i t in c re a s e d was u s u a lly le s s , and th e p oin t a t which th e in c re a s e commenced was o fte n s li g h t l y more n e g a tiv e th an f o r th e resp o n se to\ dopamine.

At p o te n tia ls more p o s itiv e th an th e r e s tin g p o te n tia l, th e curve took a s im ila r shape to th a t f o r dopamine. There was an in c re a s e in th e m agnitude o f th e inw ard c u rre n t a t p o te n tia ls between -40 mV and -20 raV, th e resp o n se reac h ed a maximum a t around -20 mV, and th e n re v e rse d , a t around 0 mV, to be seen as a n e t outw ard c u rre n t. T his s itu a tion was sim ila r to t h a t fo r dopamine, b u t, a g ain , th e o v e ra ll m agnitudes of th e induced c u r r e n ts were g e n e ra lly sm a lle r.

3 ,2 .2 .3 The Response to A c e ty lc h o lin e

Under c u rre n t-c la m p c o n dition s , a c e ty lc h o lin e a p p lie d by p re s s u re - p u lse (o r in some c a s e s by ion to p h o re sis ) caused a la rg e d e p o la ris a tion , up to 20 mV in am plitude, a s s o c ia te d w ith a marked

f a l l in th e in p u t r e sis tan c e o f th e c e l l , o fte n to a s li t t l e a s 20% of th e o r ig in a l v alu e (Figure 30), The membrane, conductance m odulated by a c e ty lc h o lin e showed no v o ltag e-d ep en d en cy , th e in c re a s e

in response siz e w ith p ro g re s s iv e h y p e rp o la ris a tion bein g alm ost lin e a r , a s p re dic te d i f th e resp on se i s m ediated by ch an n e ls which a re not v o lta g e - s e n sitiv e (F ig u re 31). By e x tra p o la tion , th e re v e rs a l p o te n tia l was e s tim a te d to be around -40 mV (mean value -44

.± SD

mV, n = ôj).

Under v o ltag e-clam p , th e resp o n se to p re s s u re -a p p lie d a c e ty lc h o lin e , a t p o te n tia ls c lo s e to th e normal r e s tin g p o te n tia l of th e c e l l, was seen a s an inw ard c u rre n t (Fig u re 3 2 ), th e m agnitude o f which was s u b s tan tia lly la r g e r th an th a t o f th e e q uiv a len t dopamine-induced c u rre n t (around 6 nA compared with 0 .5 nA fo r dopamine). As th e c e l l was step p ed to p ro g re s siv e ly more n e g a tiv e command p o te n tia ls , th e magnitude o f th e c u rren t in c re a s e d in a more o r le s s lin e a r fa shion . C onversely, a s th e membrane p o te n tia l was ste p p e d p o s itiv e , th e magnitude of th e c u rren t f e l l , and th e resp o n se re v e rs e d a t a p o te n tia l o f around -40 mV, co n firm in g th e r e s u l t s o b tain e d under cu rre n t-c la m p c o n dition s (Fig u re 33).

20mV

Figure 30 A single current-clamp response to pressure-applied acetylcholine, Resting potential was -63 mV, and current pulses were applied through a second microelectrode,

Response magnitude (mV)

Membrane potential (mV)

Figure 31 The voltage-dependence of the current-clamp response to acetylcholine, Resting potential was -59 mV, and the membrane potential was stepped to a range of values by the passage of current through a second microelectrode,

2-5 nA

Figure 32 A single voltage-clamp response to pressure-applied acetylcholine, Resting potential was -60 mV,

Current (nA)

Command Potential (mV)

Figure 33 The voltage-dependence of the voltage-clamp response to acetylcholine, Data were obtained by the ‘short-pulse* method, and the line drawn from the results of a linear regression analysis,

3 ,2 ,3 P h arm aco lo g ical D is tin c tion Between R esponses to Dopamine, R o ra d re n a lin e , OctopaminG, & A c e ty lc h o lin e

3 .2 ,3 .1 Dif f e r en tia l E f f e c ts o f SCH 23390 & Ergotam ine on R esponses to Dopamine & N o rad ren alin e

The mammalian Di r e c e p to r an ta g o nis t SCH 23390 i s a p o ten t su p p re sso r of th e resp o n se to dopamine in t hi s p re p a ra tion . Under c u rre n t-c la m p c o n dition s , a t a c o n c e n tra tion o f 1 x 10"~^’M, SCH r e v e r sib ly sdppres'seà 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 ).

Under v o ltag e-clam p c o n dition s , th e dopamine-induced c u r r e n ts were su p p ressed co m p lete ly a t a c on c e n tra tion of 1 x 10“^M, in dic a tin g a t o t a l blockade o f th e re sp 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 ls , where th e re sp on se c u rre 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 ex p erim en t (Figure 35), 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 of -160 raV, 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 se v a lu e s were re d uced 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 r e s to r e d , giv in g resp o n se c u rre n t m agnitudes of -10

nA a t -160 mV, - 0 .5 nA a t -60 mV, and +10 nA a t +30 mV. SCH 23390 had no ap p a re n t e f f e c t 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 .

C onversely, 1 x 10“ ® M SCH 23390 had no signif i c an t e f f e c t on th e c u rre n t-c la m p resp o n se to n o ra d re n a lin e , and th e m agnitude o f th e

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