Pfliigers Arch (1992) 422:129-142
Eti [ in Journal of Physiology 9 Springer-Verlag 1992
Contribution of Ca 2 + inflow to quantal, phasic transmitter release from nerve terminals of frog muscle J. Dudel Physiologisches Institut der Technischen Universit~t Mtinchen, Biedersteiner Strasse 29, W-8000 Miinchen 40, Federal Republic of Germany Received November 25, 1991/Received after revision June 22, 1992/Accepted July 13, 1992
Abstract. Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca 2+ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cde2§ or with an "intracellular", EGTA-buffered solution containing less than 0.1 pM Ca 2+ . Pulse application and recording were by a perfused macro-patchclamp electrode. The muscle outside the electrode (bath) was superfused with Ringer s solutions containing Cd 2b+ to block Ca 2+ inflow and normal (1.8 mM) or elevated (10 mM) Ca 2+ . The depolarization level of the terminal during current pulses that generated maximal Ca 2+ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca 2+ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca 2+ inflow is high. However, for large depolarizations ( > 1.5 relative units), the depression of release by block of Ca 2+ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca 2+ inflow. If the extra-electrode superfusion of Ca 2+ of the muscle was elevated to 10 mM and Cd 2+ was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1 g M C a e2+ raised the average release paradoxically. With 0.5 mM Cd 2+ this paradoxical increase of release was, on average, 4-fold at 6~ and 19-fold at 16~ Quantal endplate currents recorded in less than 0.1 ~tM Ca~ + had slightly increased amplitudes, and decay time constants were prolonged by about 50~ The results are interpreted to support the Ca 2+/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca 2+ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca 2+ concentration is sufficiently
high, large depolarizations can elicit release independent of the presence or absence of Ca 2+ inflow. Key words: Synaptic release - Ca 2+ channel block Cadmium - Depolarization-controlled release
Introduction We have shown that phasic transmitter release triggered by depolarization in the motor nerve terminal is controlled by two factors, (a) the Ca 2+ concentration in the terminal (Cai) and (b) amplitude and duration of depolarization of the membrane [8, 9, 13, 14, 22, 34, 45]. Depolarization of the membrane of the terminal usually triggers some Ca 2+ inward current, raising Cai, but in addition depolarization acts on a voltage sensor in the membrane which, by altering its conformation directly or by liberating a short-lived intracellular factor, controls the timing and partly the amount of release. The present study tries to estimate the contribution of Ca 2+ inflow to the control of release at different membrane potentials by measuring the effects of blocking or preventing Ca 2* inflow. The most convincing method to prevent Ca z+ inflow on depolarization is to remove extracellular Ca 2§ . This blocks phasic release, and this fact was the basis for the Ca 2+ theory of control of quantal transmitter release [24]. One of the effects of 0 extracellular Ca 2+ is a reduction of the resting Ca 2+, (Cai2r+). If Ca~r+ is maintained at a sufficiently high level, phasic release can be elicited even in the absence of extracellular Ca 2§ [9, 22]. One way to maintain a sufficient level of Cai is to use a perfused macro-patch electrode [9]. The ionic concentrations in a space around a section of the terminal contained in the electrode can be controlled separately from the surroundings of the rest of the terminal. If Ca 2+ outside the electrode (Ca~ § is normal or elevated, release is not blocked when Ca ~§ inflow into the terminal within the electrode is prevented by perfusion of 0 Cae2§ , or by
130 C d 2+ a d d e d to thezp+erfusate [9, 11, 12, 14]. It s e e m s t h a t e x t r a - e l e c t r o d e Cair+ c o m m u n i c a t e s w i t h i n t r a - e l e c t r o d e C a ~ +. S u c h e x p e r i m e n t s are o p e n to t h e c r i t i c i s m t h a t CaZ+-containing solution from outside the electrode m a y h a v e l e a k e d to p a r t s o f t h e t e r m i n a l w i t h t h e electrode, c o n t r i b u t i n g t o t h e r e c o r d e d release. T h i s possibility s e e m s to b e e x c l u d e d for t e c h n i c a l reasons. T h e f l u i d v o l u m e a r o u n d t h e t e r m i n a l w i t h i n t h e e l e c t r o d e was exc h a n g e d a b o u t 1000 t i m e s / s , a n d t h e f l u i d p r e s s u r e w i t h i n t h e e l e c t r o d e was set h i g h e r t h a n o u t s i d e t h e electrode, o p p o s i n g a f l o w i n t o t h e e l e c t r o d e tip. I n t h e p r e s e n t study, as a f u r t h e r i n s u r a n c e a g a i n s t c o n t r i b u t i o n s to release f r o m i n f l u e n c e s o u t s i d e t h e electrode, C d 2+ was a d d e d to t h e s u p e r f u s a t e o f t h e b a t h in c o n c e n t r a t i o n s t h a t were s u f f i c i e n t to b l o c k d e p o l a r i z a t i o n - i n d u c e d C a 2+ i n w a r d currents. U n d e r t h e s e c o n d i t i o n s t h e c o n t r i b u t i o n s o f C a 2+ i n f l o w to release in diff e r e n t r a n g e s o f d e p o l a r i z a t i o n b e c a m e o b v i o u s , A prel i m i n a r y p u b l i c a t i o n o f p a r t o f t h e results is c o n t a i n e d in [11].
Materials and methods Quantal currents were recorded from portions of endplates of excised cutaneus pectoris muscles of frogs (Ranaesculenta). A perfused macropatch-clamp electrode [11] was employed for the recording and the application of depolarizing pulses. The same electrode, with a 20-gin opening of the tip, that was used for experiments previously described [9, 14] was also employed for the present study. Recording conditions and evaluations of results were exactly as in [14]. The basic Ringer's solution contained 120 mM Na +, 2.5 mM K+, 1.8 mM Ca 2+, 126 mM CI-, 5 mM TRIS/maleate buffer and 5 mM glucose; pH 7.2. This solution is often referred to as 2 mM Ca solution. The perfusate of the electrode contained 0.2 gM tetrodotoxin (TTX) in order to prevent local excitations. In the "0 Cae" experiments, an "intracellular Ca 2+ concentration", as used for excised patches, was superfused extracellularly. Ca 2+ in the Ringer's solution was decreased to 0.2 mM, and 2 mM EGTA was added resulting in an effective free Ca 2+ concentration of less than 0.1 ~tM. After setting up the preparation and the perfusion of the electrode, the preparations were superfused with Ringer's solution for several hours, with the electrode tip in the bath and the amplifiers connected. This stabilized the preparation and electrode. When recording from a terminal, changes of the perfusate had an almost complete effect on release within a few seconds. To allow for possible slow adjustments, recording started 3 - 5 rain after a change in the perfusate. For the evaluation of amplitudes, decay time constants and delays of quantal currents, the data were evaluated offqine by a computer program, using PCM video-tape recordings (Sony PCM-501 ES), which were digitized at 50 kHz, and a HP 9817 microcomputer. To evaluate the decay time constants of the quantal currents, the program defined zero current and found the logarithm of the time course of the current. The decays then were fitted linearly.
Macro-patch-clamprecording. Since the recording and stimulating conditions are important for the validity of the data, the respective discussions given before [8, 14, 34, 35] will be summarized. The macro-electrode was a glass capillary with a 20-~tm opening, containing connections to a current-clamp stimulator and a current-clamp input. The opening of the capillary was placed on top of a nerve terminal forming a seal between the capillary and muscle fibre with about 200 k~ resistance. The distributions of current flow and voltage drop with a similar electrode were analysed by Katz and Miledi [25], Fig. 1. They showed that current passed from the interior of the electrode to the bath caused a voltage drop across the seal resistance, which in the case of negative current shifted the potential within the electrode, and around the nerve
terminal within the electrode, towards negative values. This shift in the outside potential decreased the potential difference across the membrane of the nerve terminal within the electrode. Katz and Miledi [25] used 2-txm, broken electrode tips and higher local current densities than in the present study. In their experiments the membrane resistance of the muscle or of the nerve terminal tended to break down when large depolarization currents were employed, and therefore the method was of limited usefulness. The electrodes that 1 have prepared are larger and have very smooth tips. With these electrodes, the membranes "break down" very rarely and only if excessive pressure is exerted. Stimulations and recordings are stable for many hours (see e. g. [91, Fig. 3). The seal resistance across the contact of the rim of the electrode with the muscle corresponds to a proportional drop in the intra-electrode field potential, which is generated by a current pulse. Consequently, when the intra-electrode potential is decreased above the threshold for release in proportion to a rising amplitude of current pulses, the rim area will contribute more and more to the release, and this effect will distort the measured depolarization/release relation. However, owing to the steep rise of release in the threshold region, the possible "rim release" should be relatively insignificant (see Discussion, Fig. 9). This discussion of the rim effect assumes that over all the length of the approximately 1-~tm-wide terminal, which crosses the intra-electrode area, release sites for transmitter quanta are equally and finely spread. However, the morphology and exploration of release sites with stimulating and recording electrodes show that release sites are distributed unequally. When placing the electrode for recording, therefore, one selects positions in which the distribution of quantal current amplitudes is symmetrical (see [9], Fig. 7), and in which shifts of the electrode by several micrometers do not change the recordings. In such recording positions, the contribution of the rim area to the depolarization-elicited release should be negligible. It should be noted that quantal currents that are generated in the rim area will be reduced in amplitude, since the closer the current source to the bath solution outside the electrode, the larger will be the proportion of the current that flows to the ground outside the electrode and is not recorded. The macro-patch-clamp electrode that was used for depolarization and recording from a section of a terminal was perfused in the tip region, the solution in the space between tip and the perfusion tube being enchanged several thousand times per second (see Fig. 3 B, insert). The effectiveness of this perfusion will be addressed in the Discussion.
Results Block o f Ca 2+ inflow by Cd 2d A s s h o w n b e f o r e [4, 12], 2 0 - 5 0 g M C d 2+ b l o c k s t h e dep o l a r i z a t i o n - e l i c i t e d C a 2+ c u r r e n t (/ca) i n t o t h e t e r m i n a l s (see also [20, 40]). T h r o u g h o u t t h e p r e s e n t s t u d y C d 2+ was u s e d to b l o c k /Ca in r e g i o n s o u t s i d e t h e p e r f u s e d e l e c t r o d e . I n this chapter, C d 2+ was also a p p l i e d as a blocker of/Ca into the section of the terminal within the electrode, a n d t h e results will b e c o m p a r e d to t h e effects o f 0 Cae2+ in t h e n e x t s e c t i o n . T h e release rate versus t h e d e p o l a r i z i n g c u r r e n t a m p l i t u d e is s h o w n f o r o n e t e r m i n a l in Fig. i. Two d i f f e r e n t c o n d i t i o n s were a r r a n g e d in t h e b a t h o u t s i d e t h e elect r o d e : n o r m a l Ca~ + = 2 m M w i t h 20 ~tM Cd~ + ( o p e n s y m b o l s ) , a n d h i g h Ca~ + = 10 m M w i t h 100 ~ M Cd~ + (filled s y m b o l s ) . To a c h i e v e t h e s a m e b l o c k i n g e f f e c t o n /Ca, t h e C d 2+ c o n c e n t r a t i o n has to be raised in p r o p o r t i o n to t h e C a 2+ c o n c e n t r a t i o n [1, 4]. I n t h e c o n t r o l s , with normal Ringer's solution (2mMCa~ + + TTX) p e r f u s i n g t h e e l e c t r o d e a n d 2 m M Ca~ + + 20 g M Cd~ + s u p e r f u s i n g t h e b a t h [14], t h e release rate r o s e steeply with increasing depolarization between -0.3 ~A and - 0 . 5 ~ A a n d a p p r o a c h e d a s a t u r a t i o n level o f a b o u t 0.5
131
quanta/pulse for larger depolarizations. A very similar depolarization/release relation was also observed with high Ca 2+ in the bath (10 mM Ca~ + + 100 gM Cd2+). Only the saturation level of release was elevated from 0.5 to about 7 quanta/pulse. This increase of the saturation level of release in high Ca 2+ should reflect a rise in the resting intracellular Ca 2+ concentration, Cai2r+ [9, 10, 121, which raises the effectiveness of depolarization in triggering release. The amplitudes of the depolarizing currents in the abscissae of Fig. 1 translate into depolarization voltages if the currents are multiplied by the seal resistance (see Materials and methods), which was about 200 kf~ with the electrode used here. This corresponds to a threshold for release at about 50 mV depolarization and an approach to saturation of release above about 100 mV depolarization. Another way to estimate the potential equivalents of the
10
releaserate, m quanta /pulse
depolarizing currents is the measurement of the depolarization dependence of double-pulse facilitation, F o The procedure is sketched in the inset of Fig. 1B (see also [13, 14]). F c, in relation to depolarization, is low, e.g. near 1, for small and for large depolarizations and has a sharp peak, F c max, at a moderate depolarization of - 0 . 4 gA in Fig. 1 B. Double-pulse facilitation is presumed to be generated by "residual Ca 2+'' from the facilitating prepulse [26]. Consequently, F c max represents the maximum of C a 2+ inflow at the depolarization amplitude of the respective prepulse [11, 13, 14]. The maximum /Ca occurs generally between 0 and + 20 mV membrane potential [20]. Therefore, the depolarizing current at Fc max was measured in all experiments of this study, and this value was used as a unit of depolarization in scaling the abscissae of different experiments (Figs. 2, 4 and 6). The estimate of the depolarization voltage by means of
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Fig. 1. A. Rate of phasic release of quanta, elicited by depolarizing pulses applied to a section of a terminal, versus pulse amplitude. 1), zS, Controls with 2 m M C a 2+ Ringer's perfusing the electrode and 2 m M Ca 2b+ + 2 0 g M Cd 2b+ Ringer , s superfusing the bath. The controls were measured before and after recording the effect of changing the perfusate of the electrode to 2 r a m Ca 2+ + 10 m M Cd 2+ Ringer's (o). In the second part of the experiment, the superfusate of the bath contained 10 m M Ca 2+ and 100 ~tM Cd 2+ , and controls with 2 m M Ca 2+ ( T , i ) were taken before and after recording the effect of 2 r a M Ca 2+ +20 g M Cd 2+ ( o ) . In addition to these cations, the perfusate of the electrode always contained 0.2 g M tetrodotoxin (in all experiments). B Fixed pulse facilitation, F o versus amplitude of the prepulse. The measurement procedure for F C is sehematized in the in-
set, which in the u p p e r r o w of the two panels shows the depolarizing pre- a n d fixed pulses, and in the l o w e r r o w average synaptic currents
with the q u a n t u m content m r in the case of the prepulse, and m c in the absence of a prepulse./~c, Maximal F c (Fcma• and the depolarization level of Fcmax is indicated also in A. C Rate of release in the absence of Cd 2+ (m0cde) divided by the rate of release with 20 g M Cd 2+ added (maogMcde) , versus the amplitude of the depolarizing pulse. The ordinate values correspond to the CaZ+-inflow-dependent release factor, fAca" A, Values obtained in 2 m M Ca 2+ (with 20 p M Cd2+); i , values obtained in 10 m M Ca 2+ (with 100 ~tM Cd2+). All measurements in A - C in one site. In this and in the other figures the lines between the prints are drawn by eye and are only for illustrative purposes
132 2) was about 7 with 2 0 g M C d ~ § and 0 . 4 - 0 . 6 with 1 0 0 g M and 500 g M C d 2§ With rising Cd 2+ presumably the intracellular Cd 2§ concentration within the terminal contained in the electrode, Cdiz§ , will increase. Apparently, this higher Cd 2§ makes release increasingly independent of Ca 2§ as expected if Cd 2§ is a partial agonist of Ca 2+" The experimental procedure described in Fig. 6 was repeated in six preparations. The results were always the same; f ~ c a decreased substantially as Cd 2§ was increased, and for Cd 2§ above 100 pM release in the absence of Ca 2+ was higher than in 2 m M Ca 2+ ( f A C a < 1; compare also Figs. 5 and 8). In eight experiments with 0.5 m M Ca 2+ and 10 m M Ca 2+ in the bath, the average maximal increase in release on reducing Ca 2+ to below 0.1 ~tM was 4.2-fold (range 2- to 8-fold). These results were obtained at temperatures of about 6 ~ At 16 ~ under otherwise the same conditions, the increase was on average 19-fold (n = 4, range 10- to 34-fold). The almost 5-fold increase of the effect when the temperature was raised by 10 ~ indicates the complex mode of its generation. The increase of the release level at large depolarizations on reducing Ca 2+ to below 0.1 g M is an astonish-
ing but consistent finding. This paradoxical promotion of release by less than 0.1 ~tM Ca 2+ has to remain unexplained at present. Nevertheless, this effect provides welcome evidence for the technical validity of the "zero Ca 2§ results (see Discussion). Lengthening o f E P S C in 0 Ca e Quantal excitatory postsynaptic currents (EPSC) elicited in the absence of extracellular Ca 2§ (
Eti [ in Journal of Physiology 9 Springer-Verlag 1992
Contribution of Ca 2 + inflow to quantal, phasic transmitter release from nerve terminals of frog muscle J. Dudel Physiologisches Institut der Technischen Universit~t Mtinchen, Biedersteiner Strasse 29, W-8000 Miinchen 40, Federal Republic of Germany Received November 25, 1991/Received after revision June 22, 1992/Accepted July 13, 1992
Abstract. Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca 2+ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cde2§ or with an "intracellular", EGTA-buffered solution containing less than 0.1 pM Ca 2+ . Pulse application and recording were by a perfused macro-patchclamp electrode. The muscle outside the electrode (bath) was superfused with Ringer s solutions containing Cd 2b+ to block Ca 2+ inflow and normal (1.8 mM) or elevated (10 mM) Ca 2+ . The depolarization level of the terminal during current pulses that generated maximal Ca 2+ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca 2+ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca 2+ inflow is high. However, for large depolarizations ( > 1.5 relative units), the depression of release by block of Ca 2+ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca 2+ inflow. If the extra-electrode superfusion of Ca 2+ of the muscle was elevated to 10 mM and Cd 2+ was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1 g M C a e2+ raised the average release paradoxically. With 0.5 mM Cd 2+ this paradoxical increase of release was, on average, 4-fold at 6~ and 19-fold at 16~ Quantal endplate currents recorded in less than 0.1 ~tM Ca~ + had slightly increased amplitudes, and decay time constants were prolonged by about 50~ The results are interpreted to support the Ca 2+/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca 2+ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca 2+ concentration is sufficiently
high, large depolarizations can elicit release independent of the presence or absence of Ca 2+ inflow. Key words: Synaptic release - Ca 2+ channel block Cadmium - Depolarization-controlled release
Introduction We have shown that phasic transmitter release triggered by depolarization in the motor nerve terminal is controlled by two factors, (a) the Ca 2+ concentration in the terminal (Cai) and (b) amplitude and duration of depolarization of the membrane [8, 9, 13, 14, 22, 34, 45]. Depolarization of the membrane of the terminal usually triggers some Ca 2+ inward current, raising Cai, but in addition depolarization acts on a voltage sensor in the membrane which, by altering its conformation directly or by liberating a short-lived intracellular factor, controls the timing and partly the amount of release. The present study tries to estimate the contribution of Ca 2+ inflow to the control of release at different membrane potentials by measuring the effects of blocking or preventing Ca 2* inflow. The most convincing method to prevent Ca z+ inflow on depolarization is to remove extracellular Ca 2§ . This blocks phasic release, and this fact was the basis for the Ca 2+ theory of control of quantal transmitter release [24]. One of the effects of 0 extracellular Ca 2+ is a reduction of the resting Ca 2+, (Cai2r+). If Ca~r+ is maintained at a sufficiently high level, phasic release can be elicited even in the absence of extracellular Ca 2§ [9, 22]. One way to maintain a sufficient level of Cai is to use a perfused macro-patch electrode [9]. The ionic concentrations in a space around a section of the terminal contained in the electrode can be controlled separately from the surroundings of the rest of the terminal. If Ca 2+ outside the electrode (Ca~ § is normal or elevated, release is not blocked when Ca ~§ inflow into the terminal within the electrode is prevented by perfusion of 0 Cae2§ , or by
130 C d 2+ a d d e d to thezp+erfusate [9, 11, 12, 14]. It s e e m s t h a t e x t r a - e l e c t r o d e Cair+ c o m m u n i c a t e s w i t h i n t r a - e l e c t r o d e C a ~ +. S u c h e x p e r i m e n t s are o p e n to t h e c r i t i c i s m t h a t CaZ+-containing solution from outside the electrode m a y h a v e l e a k e d to p a r t s o f t h e t e r m i n a l w i t h t h e electrode, c o n t r i b u t i n g t o t h e r e c o r d e d release. T h i s possibility s e e m s to b e e x c l u d e d for t e c h n i c a l reasons. T h e f l u i d v o l u m e a r o u n d t h e t e r m i n a l w i t h i n t h e e l e c t r o d e was exc h a n g e d a b o u t 1000 t i m e s / s , a n d t h e f l u i d p r e s s u r e w i t h i n t h e e l e c t r o d e was set h i g h e r t h a n o u t s i d e t h e electrode, o p p o s i n g a f l o w i n t o t h e e l e c t r o d e tip. I n t h e p r e s e n t study, as a f u r t h e r i n s u r a n c e a g a i n s t c o n t r i b u t i o n s to release f r o m i n f l u e n c e s o u t s i d e t h e electrode, C d 2+ was a d d e d to t h e s u p e r f u s a t e o f t h e b a t h in c o n c e n t r a t i o n s t h a t were s u f f i c i e n t to b l o c k d e p o l a r i z a t i o n - i n d u c e d C a 2+ i n w a r d currents. U n d e r t h e s e c o n d i t i o n s t h e c o n t r i b u t i o n s o f C a 2+ i n f l o w to release in diff e r e n t r a n g e s o f d e p o l a r i z a t i o n b e c a m e o b v i o u s , A prel i m i n a r y p u b l i c a t i o n o f p a r t o f t h e results is c o n t a i n e d in [11].
Materials and methods Quantal currents were recorded from portions of endplates of excised cutaneus pectoris muscles of frogs (Ranaesculenta). A perfused macropatch-clamp electrode [11] was employed for the recording and the application of depolarizing pulses. The same electrode, with a 20-gin opening of the tip, that was used for experiments previously described [9, 14] was also employed for the present study. Recording conditions and evaluations of results were exactly as in [14]. The basic Ringer's solution contained 120 mM Na +, 2.5 mM K+, 1.8 mM Ca 2+, 126 mM CI-, 5 mM TRIS/maleate buffer and 5 mM glucose; pH 7.2. This solution is often referred to as 2 mM Ca solution. The perfusate of the electrode contained 0.2 gM tetrodotoxin (TTX) in order to prevent local excitations. In the "0 Cae" experiments, an "intracellular Ca 2+ concentration", as used for excised patches, was superfused extracellularly. Ca 2+ in the Ringer's solution was decreased to 0.2 mM, and 2 mM EGTA was added resulting in an effective free Ca 2+ concentration of less than 0.1 ~tM. After setting up the preparation and the perfusion of the electrode, the preparations were superfused with Ringer's solution for several hours, with the electrode tip in the bath and the amplifiers connected. This stabilized the preparation and electrode. When recording from a terminal, changes of the perfusate had an almost complete effect on release within a few seconds. To allow for possible slow adjustments, recording started 3 - 5 rain after a change in the perfusate. For the evaluation of amplitudes, decay time constants and delays of quantal currents, the data were evaluated offqine by a computer program, using PCM video-tape recordings (Sony PCM-501 ES), which were digitized at 50 kHz, and a HP 9817 microcomputer. To evaluate the decay time constants of the quantal currents, the program defined zero current and found the logarithm of the time course of the current. The decays then were fitted linearly.
Macro-patch-clamprecording. Since the recording and stimulating conditions are important for the validity of the data, the respective discussions given before [8, 14, 34, 35] will be summarized. The macro-electrode was a glass capillary with a 20-~tm opening, containing connections to a current-clamp stimulator and a current-clamp input. The opening of the capillary was placed on top of a nerve terminal forming a seal between the capillary and muscle fibre with about 200 k~ resistance. The distributions of current flow and voltage drop with a similar electrode were analysed by Katz and Miledi [25], Fig. 1. They showed that current passed from the interior of the electrode to the bath caused a voltage drop across the seal resistance, which in the case of negative current shifted the potential within the electrode, and around the nerve
terminal within the electrode, towards negative values. This shift in the outside potential decreased the potential difference across the membrane of the nerve terminal within the electrode. Katz and Miledi [25] used 2-txm, broken electrode tips and higher local current densities than in the present study. In their experiments the membrane resistance of the muscle or of the nerve terminal tended to break down when large depolarization currents were employed, and therefore the method was of limited usefulness. The electrodes that 1 have prepared are larger and have very smooth tips. With these electrodes, the membranes "break down" very rarely and only if excessive pressure is exerted. Stimulations and recordings are stable for many hours (see e. g. [91, Fig. 3). The seal resistance across the contact of the rim of the electrode with the muscle corresponds to a proportional drop in the intra-electrode field potential, which is generated by a current pulse. Consequently, when the intra-electrode potential is decreased above the threshold for release in proportion to a rising amplitude of current pulses, the rim area will contribute more and more to the release, and this effect will distort the measured depolarization/release relation. However, owing to the steep rise of release in the threshold region, the possible "rim release" should be relatively insignificant (see Discussion, Fig. 9). This discussion of the rim effect assumes that over all the length of the approximately 1-~tm-wide terminal, which crosses the intra-electrode area, release sites for transmitter quanta are equally and finely spread. However, the morphology and exploration of release sites with stimulating and recording electrodes show that release sites are distributed unequally. When placing the electrode for recording, therefore, one selects positions in which the distribution of quantal current amplitudes is symmetrical (see [9], Fig. 7), and in which shifts of the electrode by several micrometers do not change the recordings. In such recording positions, the contribution of the rim area to the depolarization-elicited release should be negligible. It should be noted that quantal currents that are generated in the rim area will be reduced in amplitude, since the closer the current source to the bath solution outside the electrode, the larger will be the proportion of the current that flows to the ground outside the electrode and is not recorded. The macro-patch-clamp electrode that was used for depolarization and recording from a section of a terminal was perfused in the tip region, the solution in the space between tip and the perfusion tube being enchanged several thousand times per second (see Fig. 3 B, insert). The effectiveness of this perfusion will be addressed in the Discussion.
Results Block o f Ca 2+ inflow by Cd 2d A s s h o w n b e f o r e [4, 12], 2 0 - 5 0 g M C d 2+ b l o c k s t h e dep o l a r i z a t i o n - e l i c i t e d C a 2+ c u r r e n t (/ca) i n t o t h e t e r m i n a l s (see also [20, 40]). T h r o u g h o u t t h e p r e s e n t s t u d y C d 2+ was u s e d to b l o c k /Ca in r e g i o n s o u t s i d e t h e p e r f u s e d e l e c t r o d e . I n this chapter, C d 2+ was also a p p l i e d as a blocker of/Ca into the section of the terminal within the electrode, a n d t h e results will b e c o m p a r e d to t h e effects o f 0 Cae2+ in t h e n e x t s e c t i o n . T h e release rate versus t h e d e p o l a r i z i n g c u r r e n t a m p l i t u d e is s h o w n f o r o n e t e r m i n a l in Fig. i. Two d i f f e r e n t c o n d i t i o n s were a r r a n g e d in t h e b a t h o u t s i d e t h e elect r o d e : n o r m a l Ca~ + = 2 m M w i t h 20 ~tM Cd~ + ( o p e n s y m b o l s ) , a n d h i g h Ca~ + = 10 m M w i t h 100 ~ M Cd~ + (filled s y m b o l s ) . To a c h i e v e t h e s a m e b l o c k i n g e f f e c t o n /Ca, t h e C d 2+ c o n c e n t r a t i o n has to be raised in p r o p o r t i o n to t h e C a 2+ c o n c e n t r a t i o n [1, 4]. I n t h e c o n t r o l s , with normal Ringer's solution (2mMCa~ + + TTX) p e r f u s i n g t h e e l e c t r o d e a n d 2 m M Ca~ + + 20 g M Cd~ + s u p e r f u s i n g t h e b a t h [14], t h e release rate r o s e steeply with increasing depolarization between -0.3 ~A and - 0 . 5 ~ A a n d a p p r o a c h e d a s a t u r a t i o n level o f a b o u t 0.5
131
quanta/pulse for larger depolarizations. A very similar depolarization/release relation was also observed with high Ca 2+ in the bath (10 mM Ca~ + + 100 gM Cd2+). Only the saturation level of release was elevated from 0.5 to about 7 quanta/pulse. This increase of the saturation level of release in high Ca 2+ should reflect a rise in the resting intracellular Ca 2+ concentration, Cai2r+ [9, 10, 121, which raises the effectiveness of depolarization in triggering release. The amplitudes of the depolarizing currents in the abscissae of Fig. 1 translate into depolarization voltages if the currents are multiplied by the seal resistance (see Materials and methods), which was about 200 kf~ with the electrode used here. This corresponds to a threshold for release at about 50 mV depolarization and an approach to saturation of release above about 100 mV depolarization. Another way to estimate the potential equivalents of the
10
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depolarizing currents is the measurement of the depolarization dependence of double-pulse facilitation, F o The procedure is sketched in the inset of Fig. 1B (see also [13, 14]). F c, in relation to depolarization, is low, e.g. near 1, for small and for large depolarizations and has a sharp peak, F c max, at a moderate depolarization of - 0 . 4 gA in Fig. 1 B. Double-pulse facilitation is presumed to be generated by "residual Ca 2+'' from the facilitating prepulse [26]. Consequently, F c max represents the maximum of C a 2+ inflow at the depolarization amplitude of the respective prepulse [11, 13, 14]. The maximum /Ca occurs generally between 0 and + 20 mV membrane potential [20]. Therefore, the depolarizing current at Fc max was measured in all experiments of this study, and this value was used as a unit of depolarization in scaling the abscissae of different experiments (Figs. 2, 4 and 6). The estimate of the depolarization voltage by means of
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Fig. 1. A. Rate of phasic release of quanta, elicited by depolarizing pulses applied to a section of a terminal, versus pulse amplitude. 1), zS, Controls with 2 m M C a 2+ Ringer's perfusing the electrode and 2 m M Ca 2b+ + 2 0 g M Cd 2b+ Ringer , s superfusing the bath. The controls were measured before and after recording the effect of changing the perfusate of the electrode to 2 r a m Ca 2+ + 10 m M Cd 2+ Ringer's (o). In the second part of the experiment, the superfusate of the bath contained 10 m M Ca 2+ and 100 ~tM Cd 2+ , and controls with 2 m M Ca 2+ ( T , i ) were taken before and after recording the effect of 2 r a M Ca 2+ +20 g M Cd 2+ ( o ) . In addition to these cations, the perfusate of the electrode always contained 0.2 g M tetrodotoxin (in all experiments). B Fixed pulse facilitation, F o versus amplitude of the prepulse. The measurement procedure for F C is sehematized in the in-
set, which in the u p p e r r o w of the two panels shows the depolarizing pre- a n d fixed pulses, and in the l o w e r r o w average synaptic currents
with the q u a n t u m content m r in the case of the prepulse, and m c in the absence of a prepulse./~c, Maximal F c (Fcma• and the depolarization level of Fcmax is indicated also in A. C Rate of release in the absence of Cd 2+ (m0cde) divided by the rate of release with 20 g M Cd 2+ added (maogMcde) , versus the amplitude of the depolarizing pulse. The ordinate values correspond to the CaZ+-inflow-dependent release factor, fAca" A, Values obtained in 2 m M Ca 2+ (with 20 p M Cd2+); i , values obtained in 10 m M Ca 2+ (with 100 ~tM Cd2+). All measurements in A - C in one site. In this and in the other figures the lines between the prints are drawn by eye and are only for illustrative purposes
132 2) was about 7 with 2 0 g M C d ~ § and 0 . 4 - 0 . 6 with 1 0 0 g M and 500 g M C d 2§ With rising Cd 2+ presumably the intracellular Cd 2§ concentration within the terminal contained in the electrode, Cdiz§ , will increase. Apparently, this higher Cd 2§ makes release increasingly independent of Ca 2§ as expected if Cd 2§ is a partial agonist of Ca 2+" The experimental procedure described in Fig. 6 was repeated in six preparations. The results were always the same; f ~ c a decreased substantially as Cd 2§ was increased, and for Cd 2§ above 100 pM release in the absence of Ca 2+ was higher than in 2 m M Ca 2+ ( f A C a < 1; compare also Figs. 5 and 8). In eight experiments with 0.5 m M Ca 2+ and 10 m M Ca 2+ in the bath, the average maximal increase in release on reducing Ca 2+ to below 0.1 ~tM was 4.2-fold (range 2- to 8-fold). These results were obtained at temperatures of about 6 ~ At 16 ~ under otherwise the same conditions, the increase was on average 19-fold (n = 4, range 10- to 34-fold). The almost 5-fold increase of the effect when the temperature was raised by 10 ~ indicates the complex mode of its generation. The increase of the release level at large depolarizations on reducing Ca 2+ to below 0.1 g M is an astonish-
ing but consistent finding. This paradoxical promotion of release by less than 0.1 ~tM Ca 2+ has to remain unexplained at present. Nevertheless, this effect provides welcome evidence for the technical validity of the "zero Ca 2§ results (see Discussion). Lengthening o f E P S C in 0 Ca e Quantal excitatory postsynaptic currents (EPSC) elicited in the absence of extracellular Ca 2§ (
