Brain Research, 584 (1992) 123-131 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

123

BRES 17891

Calcium currents in rat motor nerve terminals Bryan R. Hamilton and Dean O. Smith Department of Physiology, University of Wisconsin, Madison, WI 53706 (USA) (Accepted 18 February 1992)

Key words: Motoneuron; Neuromuscular junction; Calcium channel; Adenosine; Adenosine triphosphate; Dihydropyridine; Conotoxin; Synapse; Inactivation

Ca 2+ currents in response to an action potential were recorded extracellularly under non-voltage clamped conditions from rat motor nerve terminals. The Ca2+ current was blocked by Cd 2÷, Co 2÷, and Ni 2÷. A residual component that could not be blocked by inorganic cations was inhibited completely by tetrodotoxin (T'I'X). The Ca 2+ current was also moderately sensitive to the N- and L-type Ca2+ channel-blocker to-conotoxin but was insensitive to the L-type channel-specific dihydropyridines. When a fraction of the terminal K + currents was blocked by 10 mM tetraethylammonium (TEA), the Ca 2÷ current duration decreased only slightly as stimulation frequency increased from 0.5 to 20 Hz. When K ÷ currents were blocked by TEA plus 3,4-diaminopyridine (250/~M) though, the Ca 2+ current duration decreased from > 70 ms to 8-10 ms as stimulation frequency increased from 0.5 to 20 Hz. Recovery of the duration following 20-Hz stimulation occurred faster during subsequent stimulation at 0.5 Hz than at 2 Hz. ATP and ACh inhibit Ca 2÷ currents at stimulation frequencies ranging from 0.5 to 20 Hz; however, when the purinergic and cholinergic autoreceptors are blocked by theophylline (100 /~M) and pirenzipine (3 gM), respectively, the frequency-induced decrease in current duration persisted. Thus, motor nerve terminal Ca 2+ current duration is determined by stimulus repetition frequency; this appears to involve intracellular Ca 2+ accumulation, although effects secondary to variability in the time course of changes in terminal membrane potentials cannot be ruled out.

INTRODUCTION T r a n s m i t t e r r e l e a s e r e q u i r e s C a 2÷ (refs. 16, 20). Since r e l e a s e d e p e n d s o n t h e e x t r a c e l l u l a r Ca 2+ conc e n t r a t i o n 6, it has b e e n i n f e r r e d t h a t t h e n e c e s s a r y Ca 2+ m u s t e n t e r t h e cell in r e s p o n s e to m e m b r a n e d e p o l a r i z a t i o n by an a c t i o n p o t e n t i a l . C a l c i u m i n w a r d c u r r e n t s have, i n d e e d , b e e n shown to flow t h r o u g h v o l t a g e - d e p e n d e n t c h a n n e l s in p r e s y n a p t i c e n d i n g s of, for e x a m p l e , t h e squid g i a n t s y n a p s e 21, t h e frog 26, t h e chick 34'38, t h e m o u s e 3,27,37, a n d t h e rat ~5. M o r e o v e r , specific Ca 2+ c h a n n e l t y p e s m e d i a t i n g t r a n s m i t t e r rel e a s e h a v e b e e n i d e n t i f i e d . T h e y i n c l u d e N - t y p e C a 2+ c h a n n e l s in c u l t u r e d rat s u p e r i o r cervical g a n g l i o n neurons ~3 a n d ' frog 26 a n d r a t cortical s y n a p t o s o m e s 35, Lt y p e c h a n n e l s in lizards t9, a n d o t h e r n e u r o n - s p e c i f i c types 1,22. N e u r o n a ! C a 2÷ c u r r e n t s m a y d e c r e a s e d u r i n g r e p e t itive c h a n n e l activation. I n molluscs, for e x a m p l e , C a 2 ÷

c h a n n e l s inactivate as i n t r a c e l l u l a r C a 2 + a c c u m u l a t e s 8. Likewise, C a 2 + - d e p e n d e n t inactivation o f N- a n d L - t y p e C a 2+ c h a n n e l s occurs in rat sensory n e u r o n s 7. This i n a c t i v a t i o n in s e n s o r y n e u r o n s a p p e a r s to b e " u s e - d e p e n d e n t " , for it is a c c e l e r a t e d as s t i m u l a t i o n f r e q u e n c y i n c r e a s e s f r o m 0.2 to 1 Hz. T h e d u r a t i o n o f m o u s e m o t o r n e r v e - t e r m i n a l Ca 2+ c u r r e n t s also d e c r e a s e s f r o m n e a r l y 350 ms to 6 ms as t h e s t i m u l a t i o n freq u e n c y i n c r e a s e s f r o m 1 / m i n to 1 0 / s 27. A l t h o u g h t h e p r e c i s e basis is unclear, this f r e q u e n c y - d e p e n d e n t d e c r e a s e m a y b e d u e to C a 2+ c h a n n e l inactivation or to activation o f a C a 2 + - d e p e n d e n t K + o r C I - c o n d u c tance. Thus, Ca 2÷ c u r r e n t s in m a m m a l i a n m o t o r nerve t e r m i n a l s have now b e e n well d e s c r i b e d , b u t t h e r e is l i m i t e d i n f o r m a t i o n a b o u t t h e sensitivity of t h e s e currents to r e p e t i t i v e stimulation. T h e goal of this study was, t h e r e f o r e , to analyze t h e f r e q u e n c y - d e p e n d e n c e o f Ca 2+ c u r r e n t s e v o k e d by r e p e t i t i v e action p o t e n t i a l s .

Correspondence: D.O. Smith, Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA. Fax: (1) (608) 262-2327.

124 T h e c u r r e n t a m p l i t u d e a n d d u r a t i o n are quite d e p e n d e n t o n nerve s t i m u l a t i o n frequency, a n d this is likely related to a c c u m u l a t i o n of i n t r a c e l l u l a r Ca 2+.

a n t a g o n i s t d T C ( 4 - 1 0 / x M ) , which e l i m i n a t e d twitching a n d c o n t a m i n a t i n g i n t e r f e r e n c e from e n d - p l a t e currents. Since d T C may affect presynaptic c u r r e n t s 1., a - b u n g a r o t o x i n was also used in some experiments.

MATERIALS AND METHODS

Experimental preparation All experiments were performed on the motor nerve terminals innervating the extensor digitorum longus (EDL) muscle of adult rats. Rats were anesthetized with chloral hydrate (2.8 mmol/kg, i.p.), and the EDL muscle along with its branch of the common peroneal nerve were carefully dissected avoiding any damage to superficial fibers and their associated nerve terminals. Care was also taken in dissecting a clean, sufficient length ( > 2 cm) of the nerve trunk for external stimulation with a suction electrode. Following dissection, the nerve-EDL preparation was pinned out at resting length in a small chamber containing 5 ml of a modified Ringer's solution comprised of the following constituents (in mM): NaCI 137, KCI 5.0, CaCI2 2.0, MgCI2 1.0, NaH2PO a 1, NaHCO 3 24, glucose 11 (pH 7.3). In experiments utilizing Cd 2+ the bicarbonate buffering system was replaced with a HEPES (10 mM) system to avoid precipitation of CdCO3; this change in buffering system did not have any independent effect on the recorded responses. A reservoir of this bathing solution was maintained at room temperature (22°C) and aerated with 95% O 2/5% CO2; this solution circulated over the preparation at 10 ml/min. Oxygen saturation was maintained at > 80% (608 Torr). The motor nerve terminals were visualized for electrode placement by adding a 10- to 20-p~M solution of the flourescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-di2ASP, Molecular Probes, Eugene, OR, USA) to the bath for 2 minu. The dye was then rinsed away during recording. Electrophysiology Focal extracellular recording techniques were identical to those described in detail in Hamilton and Smith11. Total charge recorded by the electrode was determined by integration over the area bounded by the current trace and the baseline, assuming a divalent cation as the charge carrier. Since some current is not detected by the electrode (due to the MO seal resistance), these measures of total current and total charge are underestimates of the charge crossing the membrane. Unless otherwise indicated, individual records represent averages of 30 or 50 signals. Tetrodotoxin was administered focally to the recording region from a second pipette via pressure ejection (Pneumatic Picopump, World Precision Instruments, Inc., New Haven, CT, USA). Glass pipettes (1 mm) were pulled and forged in a similar fashion to that used for the recording electrodes. Through manipulation of delivery pressure and duration of a timed pulse, variable pipettes could be calibrated to eject similar volumes (1-10 nl). Two-sided t-tests were used to determine statistical significance.

Similar results were o b t a i n e d with both c o m p o u n d s . R e c o r d s illustrating c u r r e n t s o b t a i n e d before a n d after blockage of the e n d - p l a t e c u r r e n t s are p r e s e n t e d in Fig. 1. Following complete inhibition of post-junctional electrical activity (Fig. 1A), there is an initial u p w a r d deflection indicative of an o u t w a r d c u r r e n t (Ic). This can be a t t r i b u t e d to the capacitative discharge of the t e r m i n a l m e m b r a n e by invading Na ÷ c u r r e n t from the last n o d a l region of the axon. It is insensitive to k n o w n Ca 2÷ a n d K ÷ c h a n n e l blockers, a n d if it is e l i m i n a t e d by focally applied T T X , all v o l t a g e - d e p e n d e n t activity distal to the site of T T X application is also e l i m i n a t e d . F u r t h e r m o r e , the nerve t e r m i n a l recordings of I c do not vary in m a g n i t u d e w h e n the stimulus intensity is altered at the suction electrode; they display a n " a l l - o r - n o t h i n g " response. A second o u t w a r d c u r r e n t (IK), following the capacitative c u r r e n t in time, is blocked by T E A (Fig. 1B). Since T E A is k n o w n to block an initial fast o u t w a r d K ÷ c u r r e n t a n d a C a 2 + - d e p e n d e n t K ÷ c u r r e n t at m a m m a l i a n m o t o r nerve t e r m i n a l s 27'36, this is p r e s u m a b l y a p o t a s s i u m o u t w a r d current. In the p r e s e n c e of 10 m M T E A , K ÷ blockade was c o m p l e t e within 6 min. I n all s u b s e q u e n t experiments, 10 m M T E A was used, a n d at least 10 m i n were allowed to e n s u r e full effect of the drug. A n u n d e r l y i n g inward c u r r e n t was u n c o v e r e d following blockage of the K ÷ c u r r e n t (Fig. 1B). At a 2-Hz s t i m u l a t i o n f r e q u e n c y this c u r r e n t r a n g e d from 10 to 50 p A in m a g n i t u d e a n d 8 to 10 ms in d u r a t i o n . A d d i t i o n of T E A plus a second K ÷ c h a n n e l blocker, 3 , 4 - D A P ( 2 5 0 / x M ) , revealed a n inward c u r r e n t of m u c h longer d u r a t i o n (Fig. 1C). A similar effect of b o t h T E A a n d 3 , 4 - D A P was observed in voltage recordings from the p e r i n e u r a l sheath in m o u s e m o t o r nerves 27, reflecting the p r e s e n c e of at least two K ÷ c h a n n e l types 36. Fur-

Chemicals Curare (d-tubocurarine, dTC), tetrodotoxin (TTX), tetraethylammonium chloride (TEA), nifedipine, apamin, and 3,4-diaminopyridine (3,4-DAP) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). co-conotoxinGVIA was purchased from Peninsula Laboratories, Inc. (Belmont, CA, USA). Charybdotoxin was kindly provided by C. Miller. Nitrendipine and Bay K8644 were both kindly donated by Alexander Scriabine at Miles Laboratories, Inc. (New Haven, CT).

ther a d d i t i o n of a p a m i n (10 IzM) or charybdotoxin (50 nM), which block Ca2+-activated K ÷ channels, had n o

RESULTS

the a d d i t i o n of T E A plus 3,4-DAP. T h e inward c u r r e n t is quite probably a Ca 2+ current. Since the t e r m i n a l s c a n n o t be voltage clamped, it would be i m p r u d e n t to c o n c l u d e that the entire n e t c u r r e n t is carried necessarily by Ca 2+. Alternatively,

Nerve terminal currents Nerve t e r m i n a l c u r r e n t s were r e c o r d e d from E D L muscles in the p r e s e n c e of the nicotinic A C h r e c e p t o r

effect. A n inflection in the inward c u r r e n t trace, indicated by the arrow in Fig. 1C, was often seen. It a p p e a r s to r e p r e s e n t a t r a n s i t i o n from an early s h o r t - d u r a t i o n c o m p o n e n t of the inward c u r r e n t exposed by T E A alone to a later l o n g - d u r a t i o n c o m p o n e n t revealed by

125 the current could be due to a prolonged depolarisation of the terminal due to slowed action potential repolarization, for example; in this context, the inflection might be in the repolarization phase. These possibilities were investigated and ruled highly unlikely in the following series of experiments.

A

1

Presynaptic calcium currents Dependence

on extracellular calcium. Evidence along several lines indicates that the inward current exposed following addition of TEA or both TEA and 3,4-DAP is carried by Ca *+ . In 9 experiments, such as those shown in Fig. 2A, the magnitude of the inward current was observed to increase in association with increases in extracellular Ca*+ concentration. Total charge carried by the current saturates at 3 mM (Fig. 2B), above the physiologic extracellular Ca*+ concentration (2 mM). Ba*+, too, carried the current (inset, Fig. 2B). Sensitivity to inorganic cations. The inward current was sensitive to the inorganic Ca*+-channel blockers Cd*+, Co*+, and Ni *+ (Fig. 3). Addition of Cd*+ (1 mM; n = 20) and Co*+ (10 mM; n = 4) blocked between 78 and 95% of the inward current. In similar recordings from mouse motor nerve endings, Ca*+ currents were

Fig. 1. Focal extracellular recordings of nerve terminal currents. Stimulation frequency was 0.5 Hz. A: the records seen before and after blockage of the end-plate currents (EPC) in underlying muscle by addition of o-tubocurarine (d-TC; 4 PM) to the bath. The initial upward deflection (1,) is an outward capacitative current resulting from membrane depolarization due to the influx of Na+ ions at nodes of Ranvier proximal to the terminal. The second upward deflection (Ix) represents the outward K+ ionic current. B: increasing concentrations (2.5-10.0 mM) of the K+ channel blocker TEA progressively block this outward Kf current exposing an inward terminal current. C: addition of TEA plus a second type of K+ channel blocker, 3,4-DAP (250 PM), inhibits a late, slow K+ current causing a large increase in the inward current duration. An inflection in the current (large arrow) was often seen.

ms

I

B

Y

Barium

Fig. 2. Inward current dependence on extracellular divalent cation concentration. Kf current was blocked by TEA. A: averaged records obtained at 2 Hz display an increase in the inward current as the extracellular [Ca”] is increased from 0.9 to 1.6 mM. B: the total charge recorded by the electrode is a function of extracellular [Ca”]. When Ba*+ is substituted for Ca*+ in the extracellular saline a similar response is observed (insert). In these cases, the % increase is calculated relative to the total charge recorded by the electrode at the minimum external divalent cation concentration tested (0.3 mM).

inhibited by comparable Cd*+ concentrations but by about lOO-fold lower Co*+ levels3’; there is no apparent technical explanation for this discrepancy, which suggests a possible species difference. Ni*+, which has been demonstrated to be more specific for T-type Ca*+ channel blockage 9,10,blocked I 60% of the inward current, even at relatively high concentrations (3 mM; n = 3). At most sites @SO%),a significant inward current remained following maximal blockade by Cd*+ (0.1 mM) or Co*+ (10 mM), indicating the existence of an inward component that might be insensitive to Cd*+ and Co*+. One possibility is the Ni*+-sensitive T-type Ca*+ current. However, 100 FM Ni*+ added by itself (n = 3) or following 100 PM Cd*+ (n = 5) did not result in further reduction of the remaining inward current. Assuming that the sensitivity of T-type channels to Ni*+ is equivalent in rat and chick DRG neurons’, these results indicate that the Cd*+- and Co*+-insensitive residual inward current is not the Ni2+-sensitive T-type Ca*+ current.

126 There may be a small population of Na + channels extending beyond the last node into the terminal region. In 3 out of 5 experiments, focally applied T T X had no effect on the terminal inward current prior to blockade of Na + inward current at more proximal nodes; the inward current would decrease, but this was associated with inhibition of the nerve action potential (seen as a decrease in the outward capacitative current, lc). However, in 2 experiments where the pressure ejection pipette was presumably placed closer to the terminal, the small amount of inward current that remained following 1 mM Cd 2+ blockade was found to be sensitive to TTX in a time-dependent manner (Fig. 4). Therefore, the recording electrode detected a Na +dependent component of the inward current in some but not all - preparations. The total charge carried by this component was generally small ( < 22%) relative to the Ca 2+ component. Sensitivity to organic compounds. The Ca 2+ current was moderately sensitive to 00-conotoxin, which is generally specific for N- and L-type Ca 2+ channels 23'28'3°'39.Maximal inhibition was obtained at oJ-conotoxin concentrations >_ 10 riM. At concentrations ranging from 10 to 50 nM, 35% of the inward Ca 2+ current was blocked (Fig. 5).

In contrast, dihydropyridines, which specifically affect the L-type Ca 2+ channels 12'18, produced no effect on the inward current. Micromolar concentrations of either the antagonists nitrendipine and nifedipine or the agonist Bay K8644, all well above minimum levels of effectiveness, did not alter the Ca 2+ current (Fig 5). Lower concentrations (0.1-1 ~ M ) were also without any effect. The effect of dihydropyridines is voltage dependent and may require a long time to act 29. Nerve terminal membrane potential could not be controlled with precision in this preparation. However, we did not observe any dihydropyridine effect in experiments lasting as long as 90 min in terminals that are most probably quite depolarized due to repetitive stimulation (20 Hz) in the presence of the two K + channel blockers, T E A and 3,4-DAP.

Sensitivity to stimulation frequency During repetitive stimulation, Ca 2+ current duration and amplitude became smaller. The magnitude of these changes appeared to depend on the amount of Ca 2+ entering the nerve terminals. Paired-pulse stimulation. A paired-pulse stimulation paradigm was used initially to test whether the Ca z+ 100

n

90 e..o

70

"-" 5 0

g

8 ms

~o e-~ o

~

~+"

40 3O

02+

2o

I-.10

--5 log

.............

--4 Blocker

--3 --2 Concentrotlon

--1 (M)

l

l)...I..o.~..f~ I 0.0Co2÷ 2 ms

Fig. 3. Effect of inorganic Ca2÷ channel blockers on inward current. Individual records demonstrate the effect of 0.1 mM Cda+ on the inward current in the presence of TEA plus 3,4-DAP (A) and TEA alone (B). The bottom two sets of traces demonstrate the ineffectiveness of 0.1 mM Ni2+ (C) and the effectiveness of 10 mM Coa+ (D); K+ current was blocked by TEA. E: concentration-response curves for Cd2+, Co2+ and Nia+ are shown. E×tracellular Ca2+ concentration was 2 mM in each experiment.

127

A

TTX TEA * 3,4DAP

TE_~A

2ms Fig. 4. The Na + channel blocker TTX blocks the small CdZ+-insensitive component of the inward current. After obtaining the maximum block by addition of 100 ~M Cd 2+ (indicated by the Cd 2+ arrowhead), "ITX was applied focally by pressure ejection. TTX blocked the remaining component of the inward current in a time-dependent manner; recordings were made at 30-s intervals. The pressure pipette contained 200 ~M T r X and a total of 50 nl were released, resulting in a total application of 10 pmol. Stimulation was at 2 Hz in the presence of 10 mM TEA.

currents change during repetitive stimulation. When only the TEA-sensitive K ÷ channels were blocked, thus limiting the amount of Ca 2÷ e n t e r i n g the terminals, the Ca 2÷ currents in response to the two pulses were essentially identical for inter-pulse intervals >__15 ms (Fig. 6A); differences were < 10%. If the interval

8

Fig. 6. Paired-pulse responses depend on the fraction of blocked K + currents. The two pulses were separated in time by 500 ms. A: the Ca 2÷ current exposed by addition of 10 mM TEA demonstrates no marked difference between the first and second pulses. B: however, the Ca 2÷ current exposed by addition of 10 mM TEA plus 250/.~M 3,4-DAP is significantly reduced in response to the second pulse.

between stimulus pulses was shorter than 15 ms, the response to the second pulse was smaller. However, this reduction in Ca 2÷ current correlated with a corresponding decrease in the amplitude o f the outward capacitative current (Ic), which indicates a reduced

A

Nitrendipine

B

I

Nifrendipine f

Elms

Nitrend.

Ca

I(I21

(DAP)

(II)

Bay K8644 Nifedipine

2ms

O)--CgTx

0

20

4-0 Percent

60 of

80

100

1 20

Control

Fig. 5. Effect of organic Ca 2+ channel blockers on the inward current. A: individual records were obtained at 2 Hz in the presence of TEA only, except the nitrendipine records which were recorded in the presence of TEA and 3,4-DAP. Concentrations were: nitrendipine, 10 #M; Bay K8644, 28/zM; and to-conotoxin (to-CgTx), 10 nM. B: summary of data from recordings made at 2-Hz stimulation in the presence of 10 mM TEA alone, except for "Nitrend. (DAP)" which was obtained at 0.5 Hz in the presence of 10 mM TEA plus 250 # M 3,4-DAP. Bars indicate standard errors, and the number of experiments are indicated in parentheses. Concentration ranges were: nitrendipine and nifedipine, 10/~M; Bay K8644, 10-50/zM; to-CgTx, 20-50 nM.

128 Na + influx. This is probably due to a reduction in terminal depolarization associated with an increased relative refractory period caused by the presence of TEA in the bath. In contrast, though, the longer Ca 2÷ current exposed by blockage of TEA- and 3,4-DAP-sensitive K + channels shortened significantly during paired-pulse stimulation. This is illustrated in Fig. 6B, where the current shortened considerably in response to the second pulse delivered as long as 500 ms after the first. Repetitive stimulation. During repetitive stimulation, more pronounced changes in amplitude and duration occurred as stimulation frequency increased (Fig. 7). In the presence of TEA, total charge recorded by the electrode decreased by about 30% as the frequency increased from 2 to 20 Hz (Fig. 7A). When both TEA and 3,4-DAP were added, allowing more Ca 2+ entry, repetitive stimulation at increasingly greater frequencies resulted in progressively shorter Ca 2+ current durations and amplitudes (Fig. 7B). At 20 Hz, the duration is similar to the value observed in the presence of TEA alone, 8-10 ms. This is the minimum duration of the terminal Ca 2+ current in the apparent absence of repolarizing K ÷ currents. The rate at which the duration decreases is demonstrated in Fig. 8. As stimulation frequency increases, the steady-state duration is achieved after fewer stimulus pulses, and the final duration is shorter (Fig. 8A). Moreover, the rate and extent of recovery following a

A

2Hz

2ms

B

Fig. 7. The effects of stimulation frequency. A: trains of stimuli were applied for 1 min in the presence of T E A only. W h e n stimulation frequency increased from 2 to 20 Hz, a significant reduction in Ca 2+ current was observed. B: K + currents have been blocked by T E A plus 3,4-DAP. The initial pulse in a 0.5-Hz train (single) is compared to averaged, steady-state Ca 2+ currents obtained at 0.5-, 2- and 20-Hz stimulation.

110

-

90

-

\

8o

" ~£

~.~ ~

0.5

Hz

50 40

t

~"

10

-

~F- . --1~ • 20.0 Hz 1

2 3 4 Sflmulefion

5

41- ~

6

7 #

0.5

11

8

Hz--R

2.0

1 2 3 Recovery

4

. . . . . .

Hz--R*covery

5 from

6

7 20

8 Hz

Fig. 8. Rates of Ca 2+ current shortening and recovery during repetitive stimulation. K ÷ currents were blocked by T E A plus 3,4°DAP. Ca 2+ current duration declines from the single control-pulse level to a steady state level for 0.5-, 2- and 20-Hz stimulation trains (left side). The right side of the graph demonstrates the pulse-by-pulse recovery at 0.5 and 2 Hz immediately following a 30-s train at 20 Hz. Control was defined as the duration of the first pulse in the 0.5 Hz train. The left-side curves were obtained from 21 different experiments, while the right-side curves were obtained from 4 different experiments. Vertical bars represent standard errors.

20-Hz train are also dependent on stimulation frequency (Fig. 8B). These results are consistent with frequency-dependent accumulation and clearance of intracellular Ca 2+.

Feedback inhibition by A T P and ACh Both adenosine, which is derived from hydrolysis of ATP 32, and ACh inhibit the Ca 2+ current via purinergie (A 1) and muscarinie (M 1) autoreceptors, respectively 11. Since greater amounts of ATP and ACh are released at higher stimulation frequencies, it is quite possible that these compounds cause the observed shortening of Ca 2+ current duration. This was tested by comparing Ca 2+ current duration during stimulation at frequencies ranging from 0.5 to 20 Hz before and after addition of purinergic and cholinergic agents. Both adenosine (50 /zM) and ACh (100 /xM) cause a similar reduction (30-40%) of the total inward Ca 2+ current at each frequency (Fig. 9). However, the frequency-induced reduction in current duration occurred both in the presence and the absence of adenosine or ACh. Even when both sets of autoreceptors were occupied by their respective antagonists, theophylline (100/zM) and pirenzipine (3/xM), which effectively block the autoreceptor-mediated inhibitory actions of extracellular adenosine and ACh H, the frequency-induced decrease in duration was observed to persist. Therefore, the frequency-dependent shortening of the Ca 2+ current cannot be explained by

129 purinergic or cholinergic feedback inhibition. However, autoreceptor feedback modulation may be involved in the amplitude reduction of the Ca 2÷ current seen at > 20 H z 11.

DISCUSSION During repetitive stimulation, the magnitude of the dihydropyridine-insensitive Ca 2+ currents in rat motor

A

B

0.5 Hz

0.5 Hz

l

5.25pA

lop^

lOms

8ms

2.0 Hz

2.0 Hz .

.20 Hz

.

.

.

.

.

.

.

.

.

.

.

.

.

.

I

.

.

.

.

.

2 0 Hz

Fig. 9. The effect of stimulation frequency on the Ca 2+ current in the presence of adenosine and ACh. K* currents were blocked by TEA plus 3,4-DAP. Adenosine (A) and ACh (B) depress the amplitude of the terminal Ca 2+ current at each of three stimulation frequencies - 0.5, 2 and 20 Hz. However, neither adenosine nor ACh appeared to influence the frequency-dependent decrease in Ca 2+ current duration.

130 nerve terminals depends on the extent of K+-channel blockage and on the stimulation frequency. The Ca 2+ current duration decreases more readily as a greater fraction of the K ÷ currents are blocked and as the stimulus repetition frequency increases. This relationship is most probably due to Ca2+-de pendent Ca 2+ channel inactivation. Increased K +channel blockage results in prolonged terminal depolarization and, therefore, in more Ca 2+ entry. Likewise, intracellular Ca 2+ accumulation may be expected to become elevated as stimulation frequency increases ~5. Since the terminals were not voltageclamped, frequency-dependent changes in terminal depolarization by the action potential (e.g. shorter action potentials), activation of an additional conductance (e.g. p u m p currents, exchanger currents), or inactivation of an additional population of K ÷ channels insensitive to the blocking agents used in this study, namely TEA, 3,4-DAP, apamin, and charybdotoxin, might also result in shortened Ca 2+ currents. These possibilities cannot be ruled out. However, the results are consistent parsimoniously with Ca 2+ accumulation and consequent Ca 2+ channel inactivation 8. Does this frequency-dependent reduction in Ca 2+ currents occur in vivo when K + currents are intact? This question cannot be answered with certainty in this preparation. However, circumstantial evidence indicates that it might. In the E D L muscle, synaptic facilitation, which has been attributed to accumulated intracellular Ca 2÷ (ref. 5), occurs with paired inter-pulse intervals up to 90 m s 31. Therefore, at stimulation frequencies greater than about 10 Hz, Ca 2÷ appears to accumulate to progressively higher concentrations within the terminals. As stimulation frequency increases, the amount of transmitter released per stimulus pulse also decreases progressively in rat neuromuscular junctions (e.g. ref. 33). This manifestation of synaptic depression could be consequent to reduced Ca 2+ currents. Two components of the nerve-terminal Ca 2+ current in mouse were observed following blockage of K ÷ channels by T E A and T E A plus 3,4-DAP by Penner and Dreyer 27 (cf. ref. 38). They based their conclusion on the differential sensitivities to stimulation frequency (as in this study), to Cd 2÷ blockade, and to the organic Ca 2+ channel blockers verapamil and diltiazem. In mouse, the long-duration component of the Ca 2÷ current was quite sensitive to Cd 2+ (10 /xM), while the initial, short-duration component was relatively insensitive (15 mM). In contrast, we observed both components to be nearly blocked by 100 p.M Cd 2÷ in rat. When we did observe a remaining inward current following Cd 2÷ inhibition, it was found to be sensitive

to focally applied TTX. Thus, it could be attributed to the presence of a Na + component 4. Indeed, there was evidence for a Na + component in 40% of the preparations. In their pioneering studies, Brigant and Mallart 3 concluded that the Na + channels were localized to the preterminal region (last node) and virtually non-existent at the terminal. Penner and Dreyer 27 saw no evidence of a TTX-sensitive inward current in their recordings. The fact that we did not see this Na ÷ component in every experiment supports the explanation that Ca 2+ and Na + channels are segregated on the terminal, with Ca 2 + channels congregated distally and the Na ÷ channels located more proximally. However, there may not be an abrupt decline to Na + channel density immediately distal to the last node, explaining the small and variable Na ÷ inward current. Therefore, the size of the electrode and the exact placement on the terminal would then determine how many of the proximally located Na ÷ channels were detected in the recording 17. At many sites, though, an inflection was observed in signals recorded at 0.5 Hz in the presence of T E A and 3,4-DAP as well as the additional K÷-channel blockers apamin and charybdotoxin. This could simply be due to incomplete blockage of some outward current. Alternatively, it could indicate a transition from a rapidly decaying population of Ca 2+ channels to one displaying slower kinetics, possibly due to variability in channel localization. For example, the synaptic-vesicle release region might be more richly endowed with Ca 2+ buffering and pumping machinery limiting the local rise in intracellular Ca 2÷ concentration compared to surrounding regions of the terminal. If the sensitivity of the terminal Ca 2+ current to stimulus frequency were the result of calcium-dependent Ca 2+ channel inactivation, channels farther from the release site may inactivate more readily as free Ca 2+ accumulates faster. The pharmacologic evidence is consistent with the presence of some N-type Ca 2 + channels. However, up to 65% of the current could not be blocked by w-conotoxin. Similar insensitivity to w-conotoxin has been reported in mouse motor nerve terminals 1. Thus, some additional channel type must also be present. In lizard and rodent terminals, dihydropyridine- and verapamilsensitive currents are present 1'2'19'27. However, neither we nor Penner and Dreyer 27 found dihydropyridines to have an effect on the terminal Ca 2+ currents, and Anderson and Harvey ~ were unable to block the verapamil-sensitive currents by ~o-conotoxin. Identification of Ca z+ channel type using strictly pharmacologic means leaves considerable room for uncertainty. However, it appears unlikely that L-type channels similar to

131 those o n n e u r o n a l cell bodies 25 are p r e s e n t o n m a m malian motor nerve terminals.

Acknowledgments. This work was supported by NIH Grant NS13600 and the Muscular Dystrophy Association.

REFERENCES 1 Anderson, A.J. and Harvey, A.L., to-Conotoxin does not block the verapamil-sensitive calcium channels at mouse motor nerve terminals, Neurosci. Lett., 82 (1987) 177-180. 2 Atchison, W.D. and O'Leary, S., BAY K 8644 increases release of acetylcholine at the murine neuromuscular junction, Brain Res., 419 (1987) 315-319. 3 Brigant, J.L. and Mallart, A., Presynaptic currents in mouse motor endings, J. Physiol., 333 (1982) 616-636. 4 Bourret, C. and Mallart, A., A single type of calcium channel in motor nerve endings isolated from the mouse, J. Physiol., 406 (1988) 197P. 5 Chariton, M.P., Smith, S.J. and Zucker, R.S., Rol.e of presynaptic calcium ions and channels in synaptic facilitation and depression at the squid giant synapse, J. Physiol., 323 (1982) 173-193. 6 Dodge, F.A. and Rahamimoff, R., Co-operative action of calcium ions in transmitter release at the neuromuscular junction, J. Physiol., 193 (1967) 419-432. 7 Dupont, J.L, Bossu, J.L. and Feltz, A., Effect of internal calcium concentration on calcium currents in rat sensory neurones, Pfliigers Arch., 406 (1986) 433-435. 8 Eckert, R. and Chad, J.E., Inactivation of Ca channels, Prog. Biophys. Molec. Biol., 44 (1984) 215-267. 9 Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones, J. Physiol., 394 (1987) 149-172. 10 Hagiwara, N., Irisawa, H. and Kameyama, M., Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells, J. Physiol.., 395 (1988) 233-253. 11 Hamilton, B.R. and Smith, D.O., Autoreceptor-mediated purinergic and cholinergic inhibition of motor nerve terminal calcium currents in rat, J. Physiol., 432 (1991) 327-341. 12 Hess, P., Lansman, J.B. and Tsien, R.W., Mechanism of ion permeation through calcium channels, Nature, 309 (1984) 453456. 13 Hirning, L.D., Fox, A.P., McCleskey, E.W., Olivera, B.M., Thayer, S.A., Miller, R.J. and Tsien, R.W., Dominant role of N-type Ca 2+ channels in evoked release of norepinephrine from sympathetic neurons, Science, 239 (1988) 57-61. 14 Hubbard, J.I. and Wilson, D.F., Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of D-tubocurarine, J. Physiol., 228 (1973) 307-325. 15 Jackson, M.B., Konnerth, A., and Augustine, G.F., Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals, Proc. Natl. Acad. Sci. USA, 88 (1991) 380-384. 16 Katz, B. and Miledi, R., A study of sypaptic transmission in the absence of nerve impulses, J. Physiol., 192 (1967) 407-436. 17 Konishi, T. and Sears, T.A., Electrical activity of mouse motor nerve terminals, Proc. R. Soc. London Set. B, 222 (1984) 115-120. 18 Lee, K.S. and Tsien, R.W., Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells, Nature, 302 (1983) 790-794. 19 Lindgren, C.A. and Moore, J.W., Identification of ionic currents at presynaptic nerve endings of the lizard, J. Physiol., 414 (1989) 201-222. 20 Llinas, R., Steinberg, I.Z. and Walton, K., Presynaptic calcium

currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate, Proc. Natl. Acad. Sci. USA, 73 (1976) 2918-2922. 21 Llinas, R., Steinberg, I.Z. and Walton, K., Presynaptic calcium currents in squid giant synapse, Biophys, Z, 33 (1981) 289-322. 22 Llinas, R., Sugimori, M., Lin, J.W. and Cherskey, B., Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison, Proc. Natl. Acad. Sci. USA, 86 (1989) 1689-1693. 23 McCleskey, E.W., Fox, A.P., Feldman, D., Cruz, L.J., Olivera, B.M., Tsien, R.W. and Yoshikami, D., to-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurones but not muscle, Proc. Natl. Acad. Sci. USA, 84 (1987) 4327-4331. 24 Morad, M., Goldman, Y.E. and Trentham, D.R., Rapid photochemical inactivation of Ca2+-antagonists shows that Ca 2+ entry directly activates contraction in frog heart, Nature, 304 (1983) 635-638. 25 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature, 316 (1985) 440-443. 26 Obaid, A.L., Flores, R. and Salzberg, B.M., Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to to-conotoxin and relatively insensitive to dihydropyridines: optical studies with and without voltagesenstive dyes, J. Gen. Physiol., 93 (1989) 715-729. 27 Penner, R. and Dreyer, F., Two different presynaptic calcium currents in mouse motor nerve terminals, Pfliigers Arch., 406 (1986) 190-197. 28 Plummer, M.R., Logothetis, D.E., and Hess, P., Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons, Neuron, 2 (1989) 1453-1463. 29 Rane, S.G., Holz, G.G. and Dunlap, K., Dihydropyridine inhibition of neuronal calcium current and substance P release, Pfliigers Arch., 409 (1987) 361-366. 30 Regan, L.J., Sah, D.W.Y. and Bean, B.P., Ca 2÷ channels in rat central and peripheral neurons: high threshold current resistant to dihydropyridine blockers and to-conotoxin, Neuron, (1991) 269-280. 31 Smith, D.O., Muscle-specific decrease in presynaptic calcium dependence and clearance during neuromuscular transmission in aged rats, J. Neurophysiol., 59 (1988) 1069-1082. 32 Smith, D.O., Sources of adenosine released during neuromuscular transmission in the rat, J. Physiol., 432 (1991) 343-354. 33 Smith, D.O. and Weiler, M.H., Acetylcholine metabolism and choline availability at the neuromuscular junction of mature adult and aged rats, J. Physiol., 383 (1987) 693-709. 34 Stanley, E.F., Single calcium channels on a cholinergic presynaptic nerve terminal, Neuron, 7 (1991) 585-591. 35 Suskiw, J.B., O'Leary, M.E., Murawsky, M.M. and Wang, T., Presynaptic calcium channels in rat cortical synaptosomes: fastkinetics of phasic calcium influx, channel inactivation and relationship to nitrendipine receptors, J. Neurosci. 6 (1986) 13491357. 36 Tabti, N., Bourret, C. and Mallart, A., Three potassium currents in mouse motor nerve terminals, Pfliigers Arch., 413 (1989) 395400. 37 Wiegand, H., Uhlig, S., Gotzsch, U. and Lohmann, H., The action of cobalt, cadmium and thallium on presynaptic currents in mouse motor nerve endings, Neurotoxicol. Teratol., 12 (1990) 313-318. 38 Yawo, H., Voltage-activated calcium currents in presynaptic nerve terminals of the chicken ciliary ganglion, J. Physiol., 428 (1990) 199-213. 39 Yeager, R.E., Yoshikami, D., Rivier, J., Cruz, L.J. and Miljanich, G.P., Transmitter release from presynaptic terminals of electric organ: inhibition by the calcium channel antagonist omega conus toxin, J. NeuroscL, 7 (1987) 2390-2396.

Calcium currents in rat motor nerve terminals.

Ca2+ currents in response to an action potential were recorded extracellularly under non-voltage clamped conditions from rat motor nerve terminals. Th...
786KB Sizes 0 Downloads 0 Views