Pflfigers Arch (1992) 421:256-261
Journal of Physiology 9 Springer-Verlag1992
Characteristics of synaptic transmission in reinnervating rat skeletal muscle Thomas M. Argentieri 1,., Simon P. Aiken i Swamy Laxminarayan 2, and Joseph J. McArdle 1 1 Department of Pharmacology and Toxicology and 2 The Academic Computing Center, New Jersey Medical School (UMDNJ), 185 South Orange Avenue, Newark, NJ 07103-2757, USA Received September 24, 1991/Receivedafter revision February 4, 1992/Accepted March 4, 1992
Abstract. Synaptic transmission, and its sensitivity to the effects of 3,4-diaminopyridine (3,4-DAP) and the phosphatase 2,3-butanedione monoxime (BDM), was examined for "crushed fiber" preparations of rat extensor digitorum longus muscle undergoing reinnervation after nerve crush. While mean quantal content (m) of endplate potentials (EPPs) was low early during reinnervation ( 1 0 - 24 days after nerve crush), elevation of temperature or extracellular calcium concentration restored rn toward normal. However, m achieved control values for reinnervating preparations exposed to 3,4-DAP. 3,4-DAP also activated quiescent motor nerve terminals: after exposure to this drug, synaptic transmission was detected as early as 8 days after nerve crush. BDM too activated quiescent regenerating motor nerve terminals and increased m to normal. It also prolonged EPP and endplate current decay, suggesting a pre-synaptic effect on the synchrony of transmitter release and/or a post-synaptic effect on the open time of acetylcholine-gated endplate channels. While the effects of temperature, extracellular calcium, 3,4-DAP, and BDM suggest that regenerating nerve terminals can mobilise a reserve of quanta, this reserve is abnormally low, since hemicholinium-3 caused rapid rundown of EPP amplitude at repetitively stimulated regenerating endplates.
Key words: Neuromuscular junction - Denervation Nerve regeneration 3,4-Diaminopyridine 2,3-Butanedione monoxime - Ion channels - Channel open time
which to study those processes involved in synaptogenesis. This is especially true for the neuromuscular junction (NMJ), where the functional events seen during reinnervation mimic to some extent those of synapse formation in the neonate. For example, reinnervating muscle fibers undergo a period of multiple innervation [15] similar to that seen in neonatal muscle [21]. Also, the nerve terminals regenerating into the denervated endplate produce endplate potentials (EPPs) of low quantal content [7, 23] just as occurs during normal ontogeny [20]. It is also known that endplate currents (EPCs) decay slowly and in a complex fashion during ontogeny [/0]. A similar stage of slow EPCs seems to occur during reinnervation of muscle, since miniature endplate potentials (MEPPs) initially decay slowly [16, 17] and we show in the present work that neurally evoked EPCs also have a prolonged decay time. In this study, the functional characteristics of regenerating NMJs have been further investigated. In particular, the effects of temperature, extracellular calcium concentration ([Ca 2 +]o), 3,4-diaminopyridine (3,4-DAP), and 2,3-butanedione monoxime (BDM) have been examined. The latter substance is known to depress contraction of skeletal muscle [19], although its effect on neuromuscular transmission was found to be one of enhancement [11]. Analysis of these treatments on synaptic transmission during reinnveration of muscle might provide insight into the steps involved in the restoration of synaptic function.
Materials and methods Data presented were obtained from the fasttwitch extensor digitorum longus muscle of adult Wistar rats (200300 g). Rats were anesthetised with diethyl ether and the left peroneal nerve was aseptically crushed approximately 12 mm from its entrance into the muscle. The wound was closed and animals were allowed to recover for varying intervals prior to ether anesthesia and excision of the reinnervating nerve-musclepreparation. Animals were then killed by overdose of the anesthetic. Under such conditions, functional reinnervation starts at 9-10 days after nerve crush [16]. For the purposes of this study the early period of reinnerAnimals and surgery.
Introduction The reinnervation of target organs that occurs after experimentally controlled nerve damage offers a system in * Present address." Berlex Laboratories, 110 East Hanover Avenue, Cedar Knolls, NJ 07927, USA Correspondence to: J. J. McArdle
257 vation is defined as 10-24 days after nerve crush. Control preparations were removed from the contralateral side and studied contemporaneously with the reinnervating side, or were removed from untreated animals.
Preparation. The excised muscle was stretched approximately 5% beyond its resting length and pinned to the bottom ofa Sylgard-lined Plexiglas chamber. This chamber was maintained at a temperature of 2 1 - 2 3 ~ (unless otherwise stated) and was perfused (10 mt/min) with a physiological solution containing (mM): NaC1 (135.0), KC1 (2.5), MgC12 (1.0), NaHCO3 (15.0), Na2HPO~ (1.0), CaClz (2.0) and D-glucose (11.0). The perfusate was saturated with an oxygen/ CO2 (95% : 5%) gas mixture to maintain a pH of 7 . 2 - 7.3. To elicit EPPs/EPCs the nerve was stimulated via silver bipolar electrodes with square pulses of 4 0 - 200 ItS duration. Ordinarily, pulse amplitude was supramaximal, although in experiments involvingmultiply innervated endplates both amplitude and duration were manipulated during the procedure. The muscle was crushed [2] to prevent twitch response to stimulation. The use of a perfusing medium low in K + (2.5 mM) has been shown to compensate for the block of neurotransmission secondary to nerve terminal depolarisation due to K + leakage from the crushed region of the muscle fibres [2].
Etectrophysiological recordings. Electrical activity at the NMJ was recorded either by a conventional single electrode intracellular technique, or by two electrode voltage clamp. In the former case, EPPs and MEPPs were recorded by means of a glass microelectrode (3 M KC1, 5 - 1 0 MQ resistance) inserted into the endplate membrane. The voltage clamp technique was used to study currents underlying the potentials: i.e. EPCs and miniature endplate currents (MEPCs). In this case, two electrodes filled with potassium acetate (3 M) were inserted into the endplate membrane at a distance of approximately 50 gm from each other. One electrode was used for passing current (resistance 1 - 2 Mr2), the other was used for recording (resistance 3 - 1 0 MQ). Electrodes were connected to a conventional voltage clamp device (Dagan 8500, Minneapolis, MN, USA, or Axoclamp2A, Axon Instruments, Foster City, CA, USA) and currents were recorded at a holding potential of either - 6 0 mV or - 6 5 mV, except in experiments where the voltage sensitivity of EPC was investigated. All data were filtered at 10 kHz. The output was digitized ( 5 - 4 0 kHz) and the decay time constant of EPCs (z) was analyzed on-line with a Hewlett-Packard Series 1000 digital computer (Piscataway, NJ, USA). Analysis of data. Digitized current data were fitted to the expression:
1,
= Io-
(1)
e -~'
where I, is the current at time t, Io is the initial current value, and c~ is the rate constant of current decay or 1/z [13]. The EPCs seen on the early days of reinnervationwhich exhibited multiple exponential decay were fitted to the following expression: /, = 11 " e -~*t + 12 e a2t "
(2)
where 11 and 12 are the initial current values for each component, and ~1 and c~zare the time constants of decay for each component. Direct estimates of mean quantal content (m) were obtained from the ratios of mean EPP amplitude to mean MEPP amplitude, or mean EPC amplitude to mean MEPC amplitude. EPP or EPC amplitude was obtained from the last 20 potentials/currents evoked by a train of 30 stimuli (1 Hz) applied to the nerve. The first ten recordings were not included in the mean in order to eliminate the influence of the initial decline in amplitude on the mean steady-state value. In general, 30 MEPPs or MEPCs were measured to estimate quantal size. However, since miniature frequency was very low on the early days of reinnervation [16] a smaller sample was taken then. The amplitudes of EPPs and MEPPs were corrected for the effects of membrane capacitance and non-linear summation, before calculation of m. The mean probability of transmitter release (p) was calculated from the following binomial expression [18]:
B
L
li,
II '!
'//
/ / /
/
/,au
= 1.8 ms
= 7.1 ms
]
50 nA
I 5 ms
Fig. 1. Typical endplate currents (EPCs) recorded from A the normal and B reinnervating extensor digitorum longus. (Holding potential - 6 0 mV, each trace shows the average from 20 individual recordings)
p = 1-
variance EPPS variance MEPPS F mean EPP x mean MEPP mean MEPP
(3)
It is important to point out that previous workers have found MEPP amplitudes to be abnormally distributed at reinnervating endplates [17, 23]. Furthermore, MEPP amplitude may not be an accurate estimate of the size of quanta which mediate functional transmission across the regenerating NMJ [7]. We were concerned about the impact of these findings on our estimates of m and p. Therefore, we evaluated MEPP amplitude in our preparation. We found that while mean MEPP amplitude was generally reduced during reinnervation, the distribution of the amplitudes was similar to control. This finding, and the fact that various postsynaptic factors which influence MEPP amplitude (e. g. membrane resistance [12]) are changing on the early days of reinnervation [16] led us to conclude that our estimates of m are reasonably valid.
Statistics. All data in the text and figures are presented as mean _+standard error of the mean (SEM). One-way analysis of variance (ANOVA) and Student's t-test were used to evaluate the differences, and P < 0.05 was regarded as significant.
Results
Quantal content O n the early days after the onset of muscle r e i n n e r v a t i o n , n e u r a l l y evoked EPPs a n d their u n d e r l y i n g c u r r e n t s (EPCs) were reduced in a m p l i t u d e a n d p r o l o n g e d in time course. E x a m p l e s o f E P C s are s h o w n in Fig. 1. It was f o u n d that the small a m p l i t u d e o f E P C s a n d EPPs recorded at the onset o f r e i n n e r v a t i o n was due, at least in part, to a r e d u c t i o n o f the m e a n n u m b e r (m) o f q u a n t a released in response to nerve s t i m u l a t i o n . Similar results have been reported b y others [7, 23]. A t 11 days after nerve crush, m was 10.7 +_ 1.0 (12 recordings f r o m 9 muscles), whilst the c o n t r o l value was 35.1 + 5.9 (12 recordings f r o m 12 muscles). These values were calculated f r o m p o t e n t i a l recordings, a n d w h e n c u r r e n t a m p l i t u d e s were used the m values were c o n s i s t e n t l y higher (e.g. 65.8 ___ 12.1 in controls, 4 recordings f r o m 4 muscles). T h e i n h e r e n t difficulties in the c a l c u l a t i o n o f m f r o m p o t e n t i a l recordings have b e e n discussed [22], a n d m a y explain the p o o r corr e l a t i o n b e t w e e n the results o b t a i n e d f r o m p o t e n t i a l s a n d currents. M o s t experiments r e p o r t e d in this p a p e r were p e r f o r m e d u s i n g b o t h techniques, a n d the results were always in agreement, a p a r t f r o m the fact t h a t baseline m values were c o n s i s t e n t l y smaller w h e n p o t e n t i a l s were being considered. It is therefore t h o u g h t t h a t the presen-
258
Effect of 3,4-DAP
90 80 r 0 0
70
~
5o
c ~
~
6O
4O
20 0
12~
22~
32~
Temperature Fig. 2. Effect of temperature on mean quantal content of endplate potentials (EPPs) recorded from the normal and reinnervating (15 and 20 days after nerve crush) extensor digitorum longus. Bars represent SEM, data are from a minimum of 18 fibers from at least 9 animals
40-
'4"
30
0
"~
20
As anticipated [9], the release of quanta from regenerating nerve terminals was enhanced by 3,4-DAP (6 gM). The effects of this K + channel blocker were apparent in three ways. Firstly, there was an increase in spontaneous transmitter release following EPPs. Secondly, m was increased to a normal value. F o r example, m (calculated from potentials) was 17.7 -t- 1.3 for a series of 18 fibers from 6 muscles examined at 1 1 - 1 4 days after nerve crush; within 30 min of exposure to 3,4-DAP, m had increased to 37.4 + 2.6 (25 fibres, 6 muscles), a value equivalent to c o n t r o l . T h e third and most dramatic effect of 3,4-DAP was to activate transmitter release f r o m otherwise nonactivated regenerating terminals. That is, during this study EPPs were not detected prior to 10 days after nerve crush; however, muscles twitched in response to nerve stimulation and a high percentage of impalements now produced EPPs at earlier times if 3,4-DAP was added to the superfusion solution. Specifically, 8 days after nerve crush the number of impalements resulting in EPPs increased from 0% to 61% with 3,4-DAP, and 9 days after nerve crush the percentage increased from 0% to 81%. This 3,4-DAP-induced activation of "quiescent" nerve terminals extended to at least 16 days after nerve crush.
e10.
Effect of B D M on EPC amplitude and decay 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Calcium concentration (raM) Fig. 3. Effect of extracellular calcium concentration on quantal content of EPPs recorded from the normal (squares) and reinnervating (triangles) extensor digitorum longus. Each point is the mean of at least 3 recordings from muscles denervated 11 - 14 days previously; bats represent SEM. Regression lines are shown
tation of data based on EPPs and MEPPs is reasonably valid. At 11 days after nerve crush, there was also a reduction inp, from the control value of 0.99 + 0.01 (12 recordings from 12 muscles) to 0.64 + 0.09 (12 recordings from 9 muscles). As reinnervation progressed, m and p gradually recovered, so that these two indicators of quantal release were not significantly different from controls by 33 and 16 days after nerve crush, respectively (ANOVA).
Effect of temperature, elevated [Ca 2 +/o In order to further characterize the release of transmitter from regenerating nerve terminals, we evaluated the effects of temperature and [Ca z +]o on m. While lowering the temperature to 12~ had little effect, elevation to 32~ caused m to increase for all preparations (Fig. 2). However, the magnitude of this increase was less for reinnervating muscle. Likewise, elevation of [Ca2+]o increased m in all preparations, although experimental values remained below the corresponding control (Fig. 3).
As was found in the previous study [11] using normal rat preparations, BDM increased the amplitude of EPCs and EPPs in reinnervating muscles. This effect seemed to be due to the release of a larger number of quanta. In four voltage clamp experiments on four normal preparations, m was calculated at 65.8 + 12.1 before BDM, and 91.8 + 11.5 after BDM (20 raM, 5 rain), an increase of 39.5%. The EPC amplitude in these experiments increased by 31.8 • 4.6%. Similar data were obtained from potential recordings. In reinnervating muscles the effect of BDM was qualitatively similar, but the increase in EPC amplitude tended to be more pronounced. Due to the increased difficulty in recording sufficient numbers of MEPCs from reinnervating endplates, in these preparations the increase in EPC amplitude alone was studied. Where MEPCs were recorded, their amplitude was very little altered by BDM, i.e. it is the number of quanta released that B D M affects. There were no discernable differences between the action of B D M at 12 days after nerve crush through to 19 days after nerve crush, and for this reason the data from 36 endplates (13 preparations) have been pooled. In these preparations, BDM (20 raM) increased the EPC amplitude by 99.9 • 13.1%. As reported for normal endplates [11], B D M increased at the reinnervating endplate. However, measurement of ~ in these experiments was complicated because B D M tended to cause a prolongation of the peak of the EPC/ EPP itself. A n example of such an EPP is shown in Fig. 4a. The alteration of ~ is normally thought of as a postsynaptic effect [13], but the shape of the EPPs produced when BDM is present suggests that BDM is also having a presynaptic effect; for instance, BDM may be
259 7 •
6
\\
2 mvI
E
v
&
5 X
s ms
\contro,
ZN
4
E
3 2
n kU
1
N
I
0
0
i Sum of
/ B
& •
1'o all
2'0
preceding EPPs (V)
Fig. 5. Rundown of mean EPP amplitude at a normal (triangles)and a regenerating (crosses, 14 days after nerve crush) neuromuscular junction of the extensor digitorum longus, stimulated at ]0 Hz in the presence of hemicholinium-3 (4 pM). The abscissa shows the sum of all preceding EPPs, in volts. Qualitatively similar results were obtained from 4 other matched reinnervating and control muscle fibers.
/
Control
f
10 m s
Fig. 4. A Typical EPPs recorded f r o m a reinnervating extensor digitorum longus muscle fiber, before and after 2,3-butanedione monoxime (BDM, 20 mM). The muscle was denervated 15 days previously. Resting membrane potential was approximately - 30 mV, and each trace shows the average of 20 individual recordings. B Example of multiple innervation being "uncovered" by BDM. The two EPC recordings are from an endplate of extensor digitorum longus muscle denervated 19 days previously. Stimulus parameters were identical before and after BDM (20 raM), but only one EPC was seen in response to stimulation before BDM, even though high intensity stimulus was used. (Holding potential --65 mV, each trace is the average of 20 recordings)
causing asynchronous release of transmitter. Such alterations obviously make estimates of z very dubious, and it was found that most EPCs/EPPs produced in the presence of BDM could not be fitted to simple exponentials. Best-fit values from 35 EPC recordings (12 muscles, 1 2 - 1 9 days after nerve crush) were 4.16 -t- 0.23 ms (control) and 16.9 _+ 2.2 ms (with 20 m M BDM). The z values of MEPCs and MEPPs were also increased by BDM.
EfJects of B D M on "'quiescent" nerve terminals In addition to increasing the amplitude of EPCs at the reinnervating endplate, in some cases B D M enabled a neurally evoked EPC to be produced at an endplate where there was previously no activity. This activation of quiescent endplates, comparable to the action of 3,4-DAP, was seen with 20 m M BDM especially at the early stages of reinnervation ( 1 0 - 1 6 days). BDM was also able to "uncover" multiple innervation at the regenerating endplate. At many endplates which appeared singly innervated, treatment with BDM revealed two or more innervations. An example of this effect is shown in Fig. 4b. Variation of the stimulation
parameters often enabled the component EPCs to be separated, as described for regenerating NMJs in the absence of BDM [I 5]. B D M (20 raM) uncovered multiple innervations in this manner 1 3 - 2 6 days after nerve crush. The phenomenon was not noted with 3,4-DAP. The complex EPCs as illustrated in Fig. 4b are not likely to result from repetitive firing of the nerve terminal, since it was noted that at some endplates the first peak would always be larger than the subsequent peak/s, while at other endplates it would always be smaller.
Spontaneous activity due to B D M A further action of BDM was apparent which was confined to the period 2 0 - 24 days after nerve crush. Application of BDM (20 raM) caused bursts of spontaneous EPC activity, at approximately 1 - 2 Hz. The frequency of the bursts gradually declined, until they had disappeared after 15 rain of contact with BDM. This effect of BDM was highly reproducible, and was only seen during this narrow time band of reinnervation.
Effect of hemicholinium-3 While the response to temperature, [Ca2+]o, 3,4-DAP, and BDM indicated that regenerating nerve terminals have a reserve of quanta enabling them to respond to appropriate stimuli with an increase of m, the reserve is less than normal. This was apparent for preparations continuously stimulated in the presence of hemicholinium-3. Stimulation of reinnervating preparations caused rapid cessation of quantal release when hemicholinium-3 was used (Fig. 5).
EPC decay during reinnervation As Fig. 1 shows, EPCs decay slowly at the beginning of functional reinnervation of muscle. Specifically, the time
260 Table 1. Effect ofneostigmine (1.0 gM) on the time constant of EPC decay in the rat extensor digitorum longus Days after nerve crush
Time constant (ms)
Normal 11 - 14
Control
Neostigmine
1.46 _+ 0.04 4.47 _+ 0.70
13.50 _+ 0.98 9.99 _+ 0.51
Data are shown as mean • SEM, 10 fibers (at least 8 muscles) for each point. Holding potential - 6 0 mV
nA 1501
loo4 &
[] 5o-l -60 -50 L~ I
-40 I
A A
[]
[]
-30 I
A []
-20 I z~ L~
- 10 I
[]
A
A
J
Z~ =
i
=
10
20
30
40
5'0 mV
-50 -100
-150
Fig. 6. Typical current/voltage relationship for the EPC amplitude of a normal (squares) and a reinnervating (triangles) endplate
constant of EPC decay (z) was 6.00 _+ 0.69 ms (12 recordings from 6 muscles) at 12 days after nerve crush while the control value was 1.51 _+ 0.02 ms (156 recordings from 41 muscles). It is unlikely that this prolongation can be explained in terms of a denervation-induced reduction of endplate cholinesterase activity, since exposure to neostigmine (i.0 gM, 5 min) significantly prolonged ~ in both control and reinnervating preparations (Table 1). In addition to this prolonged monoexponential decay, it was also found that 30% of the EPCs examined between 11 and 22 days after nerve crush followed a double exponential decay. The mean values of T for the rapid and slow phases of decay were 1.70___ 0.26 and 3.51 _+ 0.19 ms, respectively. After 22 days such complex EPC decay was no longer detected, and z gradually recovered to normal by 30 days after nerve crush. It is interesting to note that while none of the hundreds of control EPCs examined at 22~ followed a double exponential decay, they occasionally did so when the temperature was lowered to 12~C. It has been reported [8] that two Lorentzian curves are sometimes necessary to describe the power spectrum of acetylcholine-evoked noise at the innervated endplate of frog muscle at 8 11~ Although EPCs were abnormal in amplitude and time course of decay early during reinnervation they continued to exhibit normal voltage sensitivity (Fig. 6). That is, amplitude remained a linear function of the holding potential and the average reversal potential was equivalent to control ( - 3 mV in both cases). Also,just as for normal
endplates, values of ~ were less at more positive holding potentials. However, ~ was more sensitive to voltage for reinnervating endplates; i.e. at reinnervating endplates there was an e-fold change in ~ for a 45 mV change of holding potential, while in controls a similar change of occurred with a 170 mV shift of holding potential.
Discussion The results presented here are in agreement with earlier reports of low quantal release of acetylcholine from regenerating motor nerve terminals in mouse [23] and frog [7]. As in the latter tissue, elevation of temperature or [Ca 2+]o increased m for regenerating NMJs. In addition, we studied the effects of drugs capable of enhancing transmission across the regenerating NMJ. The K § channel blocker 3,4-DAP increased m to normal during the early days of reinnervation, and EPPs could be elicited at early times after nerve crush. The phosphatase BDM also increased transmitter release and enabled EPPs/ EPCs to be recorded from quiescent endplates. The action of 3,4-DAP at very early times after nerve crush is particularly interesting in view of the "non-transmitting" stage of synapse formation found in some species during reinnervation of muscle. It has previously been reported [17] that MEPP frequency and amplitude were both normal before EPPs could be recorded at regenerating NMJs in adult frog muscle. In contrast to the amphibian muscle, a "non-transmitting" stage is at best infrequent during reinnervation of mammalian muscle [6, 23], and our own work has suggested that EPPs and MEPPs reappear at the same time during the reinnervation of rat hind-limb muscle [15]. Nevertheless, our present finding that EPPs/EPCs can be elicited at early times after nerve crush when preparations are pharmacologically treated justifies extension of the hypothesis of Dennis and Miledi [6] to regenerating mammalian NMJs. 3,4-DAP is known to block K § channels in pre-synaptic nerve membrane, thereby increasing the Ca 2 § influx into motor nerve terminals [14]. The ability of 3,4-DAP to activate transmitter release early in regeneration is therefore not surprising. Our data suggest that a "non-transmitting" phase of synapse formation may be overcome by increasing Ca 2 § influx. Although the exact mechanism by which BDM facilitates transmission has not been identified, it has been suggested [11] that BDM can dephosphorylate K + channels and inactivate them, hence prolonging the nerve terminal depolarisation in response to an action potential. It appears that BDM may have both pre- and postsynaptic effects, increasing transmitter release and prolonging the mean open time of the channels associated with acetylcholine receptors. A denervation-induced reduction in cholinesterase activity cannot fully explain the prolongation of EPCs and MEPCs that we observed during reinnervation, since neostigmine prolonged ~ at both normal and regenerating NMJs (Table 1), albeit to a slightly lesser extent at the latter. This observation is comparable to that made at the developing mammalian NMJ [10]. A second analogy with
261 the d e v e l o p i n g N M J is the o c c u r r e n c e o f c o m p l e x E P C d e c a y d u r i n g r e i n n e r v a t i o n o f a d u l t r a t h i n d - l i m b muscle. These findings c a n be i n t e r p r e t e d as a d e n e r v a t i o n i n d u c e d a l t e r a t i o n o f the o p e n time o f e n d p l a t e c h a n n e l s a s s o c i a t e d with the a c e t y l c h o l i n e receptor. A l t h o u g h we f o u n d t h a t E P P a n d M E P C d e c a y was p r o l o n g e d d u r i n g r e i n n e r v a t i o n , o t h e r w o r k e r s [3] h a v e f o u n d t h a t M E P P s a n d E P P s follow a n o r m a l time c o u r s e at r e g e n e r a t i n g N M J s in r a t d i a p h r a g m . B r e n n e r a n d S a k m a n n [4] r e p o r t e d t h a t the c h a n n e l o p e n time is n o t c h a n g e d d u r i n g r e i n n e r v a t i o n . This c o n c l u s i o n was b a s e d on three successful e x p e r i m e n t s in w h i c h they a n a l y z e d the s p e c t r u m o f a c e t y l c h o l i n e - i n d u c e d e n d p l a t e noise 1 8 - 20 d a y s after d e n e r v a t i n g the r a t d i a p h r a g m . T h e r e are c o n s i d e r a b l e differences in t e c h n i q u e b e t w e e n their s t u d y a n d o u r o w n , in p a r t i c u l a r the fact t h a t we a l l o w e d the nerve t e r m i n a l itself to " a p p l y " the acetylcholine, w h i c h m i g h t be o f significance if there are several dist a n t l y l o c a t e d release sites o n the r e g e n e r a t i n g nerve term i n a l [5]. I n a d d i t i o n , a differential effect o f p e r h y d r o h i s t r i o n i c o t o x i n on E P C s p r o d u c e d b y i o n t o p h o r e t i c a l l y versus n e u r a l t y a p p l i e d a c e t y l c h o l i n e has been n o t e d [1]. T h e r e f o r e , it is p o s s i b l e t h a t differences in m e t h o d o f a p p l i c a t i o n o f a c e t y l c h o l i n e m a y c o n t r i b u t e to the difference b e t w e e n o u r f i n d i n g a n d t h a t o f B r e n n e r a n d S a k m a n n [4]. O u r o w n w o r k has c o n s i s t e n t l y s h o w n p r o l o n g a t i o n o f M E P P a n d E P P d e c a y t i m e in r a t h i n d - l i m b muscles (e. g. [16]), c o n s i s t e n t w i t h a n i n c r e a s e d c h a n n e l o p e n time. P r o l o n g a t i o n o f M E P P d e c a y has also been r e p o r t e d at the r e i n n e r v a t i n g frog N M J [17].
Acknowledgements. We thank Dr. Stanley Von Hagen for his help in the statistical and graphical presentation of the data. This work was supported by grants NS-11055-09 from the National Institute of Neurological Disorders and Stroke, and 5 R01 AA08025 from the National Institute on Alcohol Abuse and Alcoholism. References
1. Albuquerque EX, Gage PW (1978) Differential effects of perhydrohistrionicotoxin on neurally and iontophoretically evoked endplate currents. Proc Natl Acad Sci USA 75:1596-1599 2. Barstad JAB, Lilleheil G (1968) Transversely cut diaphragm preparation from rat. An adjuvant tool in the study of the physiology and pharmacology of the myoneural junction. Arch Int Pharmacodyn Ther 175 : 373 - 390 3. Bennett MR, McLachlan EM, Taylor RS (1973) The formation of synapses in reinnervated mammalian striated muscle. J Physiol (Lond) 233:481- 500
4. Brenner HR, Sakmann B (1983) Neutrotrophic control of channel properties at neuromuscular synapses of rat muscle. J Physiol (Lond) 337:159-171 5. D'Alonzo AJ, Grinnel AD (1985) Profiles of evoked release along the length of frog motor nerve terminals. J Physiol (Lond) 359:235 -258 6. Dennis MJ, Miledi R (1974) Non-transmitting neuromuscular junctions during an early stage of end-plate reinnervation. J Physiol (Lond) 239 : 553 - 570 7. Dennis M J, Miledi R (1974) Characteristics of transmitter release at regenerating frog neuromuscular junctions. J Physiol (Lond) 239 : 571 - 594 8. Dreyer F, Walther Chr, Peper K (1976) Junctional and extrajunctional acetylcholine receptors in normal and denervated frog muscle fibres. Pflfigers Arch 366:1 - 9 9. Durant NN, Marshall IG (1980) The effects of 3,4-diaminopyridine on acetylcholine release at the frog neuromuscular junction. Eur J Pharmacol 67:201- 208 10. Fischbach GD, Schuetze SM (1980) A post-natal decrease in acetylcholine channel open time at rat end-plates. J Physiol (Lond) 303:125-137 11. Gage PW, McArdle J J, Saint DA (1990) Effects of butanedione monoxime on neuromuscular transmission. Br J Pharmacol 100:467- 470 12. Katz B, Thesleff S (1957) On the factors which determine the amplitude of the 'miniature end-plate potential'. J Physiol (Lond) 137:267 -278 13. Magleby KL, Stevens CF (1972) A quantitative description of end-plate currents. J Physiol (Lond) 223:173 - 197 14. Mallart A (1985) Electric current flow inside perineural sheaths of mouse motor nerves. J Physiol (Lond) 368 : 5 6 5 - 575 15. McArdle JJ (1975) Complex end-plate potentials at the regenerating neuromuscular junction of the rat. Exp Neurol 49:629638 16. McArdle JJ, Albuquerque EX (1973) A study of the reinnervation of fast and slow mammalian muscles. J Gen Physiol 61:1 - 2 3 17. Miledi R (1960) Properties of regenerating neuromuscular synpases in the frog. J Physiol (Lond) 154:190-205 18. Miyamoto MD (1975) Binomial analysis of quantal transmitter release at glycerol treated frog neuromuscular junctions. J Physiol (Lond) 250:121 - 142 19. Mulieri LA, Alpert NR (1984) Differential effects of 2,3butanedione monoxime (BDM) on activation and contraction. Biophys J 45 : 47 a 20. Pagala MKD, Tada S, Namba T, Grob D (1982) Neuromuscular transmission in neonatal mice injected with serum globulin of myasthenia gravis patients. Neurology 32:12-17 21. Redfern PA (1970) Neuromuscular transmission in new-born rats. J Physiol (Lond) 209:701 - 7 0 9 22. Stevens CF (1976) A comment on Martin's relation. Biophys J 16:891-895 23. Tonge DA (1974) Physiological characteristics of re-innervation of skeletal muscle in the mouse. J Physiol (Lond) 241 : 141 - 153
Journal of Physiology 9 Springer-Verlag1992
Characteristics of synaptic transmission in reinnervating rat skeletal muscle Thomas M. Argentieri 1,., Simon P. Aiken i Swamy Laxminarayan 2, and Joseph J. McArdle 1 1 Department of Pharmacology and Toxicology and 2 The Academic Computing Center, New Jersey Medical School (UMDNJ), 185 South Orange Avenue, Newark, NJ 07103-2757, USA Received September 24, 1991/Receivedafter revision February 4, 1992/Accepted March 4, 1992
Abstract. Synaptic transmission, and its sensitivity to the effects of 3,4-diaminopyridine (3,4-DAP) and the phosphatase 2,3-butanedione monoxime (BDM), was examined for "crushed fiber" preparations of rat extensor digitorum longus muscle undergoing reinnervation after nerve crush. While mean quantal content (m) of endplate potentials (EPPs) was low early during reinnervation ( 1 0 - 24 days after nerve crush), elevation of temperature or extracellular calcium concentration restored rn toward normal. However, m achieved control values for reinnervating preparations exposed to 3,4-DAP. 3,4-DAP also activated quiescent motor nerve terminals: after exposure to this drug, synaptic transmission was detected as early as 8 days after nerve crush. BDM too activated quiescent regenerating motor nerve terminals and increased m to normal. It also prolonged EPP and endplate current decay, suggesting a pre-synaptic effect on the synchrony of transmitter release and/or a post-synaptic effect on the open time of acetylcholine-gated endplate channels. While the effects of temperature, extracellular calcium, 3,4-DAP, and BDM suggest that regenerating nerve terminals can mobilise a reserve of quanta, this reserve is abnormally low, since hemicholinium-3 caused rapid rundown of EPP amplitude at repetitively stimulated regenerating endplates.
Key words: Neuromuscular junction - Denervation Nerve regeneration 3,4-Diaminopyridine 2,3-Butanedione monoxime - Ion channels - Channel open time
which to study those processes involved in synaptogenesis. This is especially true for the neuromuscular junction (NMJ), where the functional events seen during reinnervation mimic to some extent those of synapse formation in the neonate. For example, reinnervating muscle fibers undergo a period of multiple innervation [15] similar to that seen in neonatal muscle [21]. Also, the nerve terminals regenerating into the denervated endplate produce endplate potentials (EPPs) of low quantal content [7, 23] just as occurs during normal ontogeny [20]. It is also known that endplate currents (EPCs) decay slowly and in a complex fashion during ontogeny [/0]. A similar stage of slow EPCs seems to occur during reinnervation of muscle, since miniature endplate potentials (MEPPs) initially decay slowly [16, 17] and we show in the present work that neurally evoked EPCs also have a prolonged decay time. In this study, the functional characteristics of regenerating NMJs have been further investigated. In particular, the effects of temperature, extracellular calcium concentration ([Ca 2 +]o), 3,4-diaminopyridine (3,4-DAP), and 2,3-butanedione monoxime (BDM) have been examined. The latter substance is known to depress contraction of skeletal muscle [19], although its effect on neuromuscular transmission was found to be one of enhancement [11]. Analysis of these treatments on synaptic transmission during reinnveration of muscle might provide insight into the steps involved in the restoration of synaptic function.
Materials and methods Data presented were obtained from the fasttwitch extensor digitorum longus muscle of adult Wistar rats (200300 g). Rats were anesthetised with diethyl ether and the left peroneal nerve was aseptically crushed approximately 12 mm from its entrance into the muscle. The wound was closed and animals were allowed to recover for varying intervals prior to ether anesthesia and excision of the reinnervating nerve-musclepreparation. Animals were then killed by overdose of the anesthetic. Under such conditions, functional reinnervation starts at 9-10 days after nerve crush [16]. For the purposes of this study the early period of reinnerAnimals and surgery.
Introduction The reinnervation of target organs that occurs after experimentally controlled nerve damage offers a system in * Present address." Berlex Laboratories, 110 East Hanover Avenue, Cedar Knolls, NJ 07927, USA Correspondence to: J. J. McArdle
257 vation is defined as 10-24 days after nerve crush. Control preparations were removed from the contralateral side and studied contemporaneously with the reinnervating side, or were removed from untreated animals.
Preparation. The excised muscle was stretched approximately 5% beyond its resting length and pinned to the bottom ofa Sylgard-lined Plexiglas chamber. This chamber was maintained at a temperature of 2 1 - 2 3 ~ (unless otherwise stated) and was perfused (10 mt/min) with a physiological solution containing (mM): NaC1 (135.0), KC1 (2.5), MgC12 (1.0), NaHCO3 (15.0), Na2HPO~ (1.0), CaClz (2.0) and D-glucose (11.0). The perfusate was saturated with an oxygen/ CO2 (95% : 5%) gas mixture to maintain a pH of 7 . 2 - 7.3. To elicit EPPs/EPCs the nerve was stimulated via silver bipolar electrodes with square pulses of 4 0 - 200 ItS duration. Ordinarily, pulse amplitude was supramaximal, although in experiments involvingmultiply innervated endplates both amplitude and duration were manipulated during the procedure. The muscle was crushed [2] to prevent twitch response to stimulation. The use of a perfusing medium low in K + (2.5 mM) has been shown to compensate for the block of neurotransmission secondary to nerve terminal depolarisation due to K + leakage from the crushed region of the muscle fibres [2].
Etectrophysiological recordings. Electrical activity at the NMJ was recorded either by a conventional single electrode intracellular technique, or by two electrode voltage clamp. In the former case, EPPs and MEPPs were recorded by means of a glass microelectrode (3 M KC1, 5 - 1 0 MQ resistance) inserted into the endplate membrane. The voltage clamp technique was used to study currents underlying the potentials: i.e. EPCs and miniature endplate currents (MEPCs). In this case, two electrodes filled with potassium acetate (3 M) were inserted into the endplate membrane at a distance of approximately 50 gm from each other. One electrode was used for passing current (resistance 1 - 2 Mr2), the other was used for recording (resistance 3 - 1 0 MQ). Electrodes were connected to a conventional voltage clamp device (Dagan 8500, Minneapolis, MN, USA, or Axoclamp2A, Axon Instruments, Foster City, CA, USA) and currents were recorded at a holding potential of either - 6 0 mV or - 6 5 mV, except in experiments where the voltage sensitivity of EPC was investigated. All data were filtered at 10 kHz. The output was digitized ( 5 - 4 0 kHz) and the decay time constant of EPCs (z) was analyzed on-line with a Hewlett-Packard Series 1000 digital computer (Piscataway, NJ, USA). Analysis of data. Digitized current data were fitted to the expression:
1,
= Io-
(1)
e -~'
where I, is the current at time t, Io is the initial current value, and c~ is the rate constant of current decay or 1/z [13]. The EPCs seen on the early days of reinnervationwhich exhibited multiple exponential decay were fitted to the following expression: /, = 11 " e -~*t + 12 e a2t "
(2)
where 11 and 12 are the initial current values for each component, and ~1 and c~zare the time constants of decay for each component. Direct estimates of mean quantal content (m) were obtained from the ratios of mean EPP amplitude to mean MEPP amplitude, or mean EPC amplitude to mean MEPC amplitude. EPP or EPC amplitude was obtained from the last 20 potentials/currents evoked by a train of 30 stimuli (1 Hz) applied to the nerve. The first ten recordings were not included in the mean in order to eliminate the influence of the initial decline in amplitude on the mean steady-state value. In general, 30 MEPPs or MEPCs were measured to estimate quantal size. However, since miniature frequency was very low on the early days of reinnervation [16] a smaller sample was taken then. The amplitudes of EPPs and MEPPs were corrected for the effects of membrane capacitance and non-linear summation, before calculation of m. The mean probability of transmitter release (p) was calculated from the following binomial expression [18]:
B
L
li,
II '!
'//
/ / /
/
/,au
= 1.8 ms
= 7.1 ms
]
50 nA
I 5 ms
Fig. 1. Typical endplate currents (EPCs) recorded from A the normal and B reinnervating extensor digitorum longus. (Holding potential - 6 0 mV, each trace shows the average from 20 individual recordings)
p = 1-
variance EPPS variance MEPPS F mean EPP x mean MEPP mean MEPP
(3)
It is important to point out that previous workers have found MEPP amplitudes to be abnormally distributed at reinnervating endplates [17, 23]. Furthermore, MEPP amplitude may not be an accurate estimate of the size of quanta which mediate functional transmission across the regenerating NMJ [7]. We were concerned about the impact of these findings on our estimates of m and p. Therefore, we evaluated MEPP amplitude in our preparation. We found that while mean MEPP amplitude was generally reduced during reinnervation, the distribution of the amplitudes was similar to control. This finding, and the fact that various postsynaptic factors which influence MEPP amplitude (e. g. membrane resistance [12]) are changing on the early days of reinnervation [16] led us to conclude that our estimates of m are reasonably valid.
Statistics. All data in the text and figures are presented as mean _+standard error of the mean (SEM). One-way analysis of variance (ANOVA) and Student's t-test were used to evaluate the differences, and P < 0.05 was regarded as significant.
Results
Quantal content O n the early days after the onset of muscle r e i n n e r v a t i o n , n e u r a l l y evoked EPPs a n d their u n d e r l y i n g c u r r e n t s (EPCs) were reduced in a m p l i t u d e a n d p r o l o n g e d in time course. E x a m p l e s o f E P C s are s h o w n in Fig. 1. It was f o u n d that the small a m p l i t u d e o f E P C s a n d EPPs recorded at the onset o f r e i n n e r v a t i o n was due, at least in part, to a r e d u c t i o n o f the m e a n n u m b e r (m) o f q u a n t a released in response to nerve s t i m u l a t i o n . Similar results have been reported b y others [7, 23]. A t 11 days after nerve crush, m was 10.7 +_ 1.0 (12 recordings f r o m 9 muscles), whilst the c o n t r o l value was 35.1 + 5.9 (12 recordings f r o m 12 muscles). These values were calculated f r o m p o t e n t i a l recordings, a n d w h e n c u r r e n t a m p l i t u d e s were used the m values were c o n s i s t e n t l y higher (e.g. 65.8 ___ 12.1 in controls, 4 recordings f r o m 4 muscles). T h e i n h e r e n t difficulties in the c a l c u l a t i o n o f m f r o m p o t e n t i a l recordings have b e e n discussed [22], a n d m a y explain the p o o r corr e l a t i o n b e t w e e n the results o b t a i n e d f r o m p o t e n t i a l s a n d currents. M o s t experiments r e p o r t e d in this p a p e r were p e r f o r m e d u s i n g b o t h techniques, a n d the results were always in agreement, a p a r t f r o m the fact t h a t baseline m values were c o n s i s t e n t l y smaller w h e n p o t e n t i a l s were being considered. It is therefore t h o u g h t t h a t the presen-
258
Effect of 3,4-DAP
90 80 r 0 0
70
~
5o
c ~
~
6O
4O
20 0
12~
22~
32~
Temperature Fig. 2. Effect of temperature on mean quantal content of endplate potentials (EPPs) recorded from the normal and reinnervating (15 and 20 days after nerve crush) extensor digitorum longus. Bars represent SEM, data are from a minimum of 18 fibers from at least 9 animals
40-
'4"
30
0
"~
20
As anticipated [9], the release of quanta from regenerating nerve terminals was enhanced by 3,4-DAP (6 gM). The effects of this K + channel blocker were apparent in three ways. Firstly, there was an increase in spontaneous transmitter release following EPPs. Secondly, m was increased to a normal value. F o r example, m (calculated from potentials) was 17.7 -t- 1.3 for a series of 18 fibers from 6 muscles examined at 1 1 - 1 4 days after nerve crush; within 30 min of exposure to 3,4-DAP, m had increased to 37.4 + 2.6 (25 fibres, 6 muscles), a value equivalent to c o n t r o l . T h e third and most dramatic effect of 3,4-DAP was to activate transmitter release f r o m otherwise nonactivated regenerating terminals. That is, during this study EPPs were not detected prior to 10 days after nerve crush; however, muscles twitched in response to nerve stimulation and a high percentage of impalements now produced EPPs at earlier times if 3,4-DAP was added to the superfusion solution. Specifically, 8 days after nerve crush the number of impalements resulting in EPPs increased from 0% to 61% with 3,4-DAP, and 9 days after nerve crush the percentage increased from 0% to 81%. This 3,4-DAP-induced activation of "quiescent" nerve terminals extended to at least 16 days after nerve crush.
e10.
Effect of B D M on EPC amplitude and decay 0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Calcium concentration (raM) Fig. 3. Effect of extracellular calcium concentration on quantal content of EPPs recorded from the normal (squares) and reinnervating (triangles) extensor digitorum longus. Each point is the mean of at least 3 recordings from muscles denervated 11 - 14 days previously; bats represent SEM. Regression lines are shown
tation of data based on EPPs and MEPPs is reasonably valid. At 11 days after nerve crush, there was also a reduction inp, from the control value of 0.99 + 0.01 (12 recordings from 12 muscles) to 0.64 + 0.09 (12 recordings from 9 muscles). As reinnervation progressed, m and p gradually recovered, so that these two indicators of quantal release were not significantly different from controls by 33 and 16 days after nerve crush, respectively (ANOVA).
Effect of temperature, elevated [Ca 2 +/o In order to further characterize the release of transmitter from regenerating nerve terminals, we evaluated the effects of temperature and [Ca z +]o on m. While lowering the temperature to 12~ had little effect, elevation to 32~ caused m to increase for all preparations (Fig. 2). However, the magnitude of this increase was less for reinnervating muscle. Likewise, elevation of [Ca2+]o increased m in all preparations, although experimental values remained below the corresponding control (Fig. 3).
As was found in the previous study [11] using normal rat preparations, BDM increased the amplitude of EPCs and EPPs in reinnervating muscles. This effect seemed to be due to the release of a larger number of quanta. In four voltage clamp experiments on four normal preparations, m was calculated at 65.8 + 12.1 before BDM, and 91.8 + 11.5 after BDM (20 raM, 5 rain), an increase of 39.5%. The EPC amplitude in these experiments increased by 31.8 • 4.6%. Similar data were obtained from potential recordings. In reinnervating muscles the effect of BDM was qualitatively similar, but the increase in EPC amplitude tended to be more pronounced. Due to the increased difficulty in recording sufficient numbers of MEPCs from reinnervating endplates, in these preparations the increase in EPC amplitude alone was studied. Where MEPCs were recorded, their amplitude was very little altered by BDM, i.e. it is the number of quanta released that B D M affects. There were no discernable differences between the action of B D M at 12 days after nerve crush through to 19 days after nerve crush, and for this reason the data from 36 endplates (13 preparations) have been pooled. In these preparations, BDM (20 raM) increased the EPC amplitude by 99.9 • 13.1%. As reported for normal endplates [11], B D M increased at the reinnervating endplate. However, measurement of ~ in these experiments was complicated because B D M tended to cause a prolongation of the peak of the EPC/ EPP itself. A n example of such an EPP is shown in Fig. 4a. The alteration of ~ is normally thought of as a postsynaptic effect [13], but the shape of the EPPs produced when BDM is present suggests that BDM is also having a presynaptic effect; for instance, BDM may be
259 7 •
6
\\
2 mvI
E
v
&
5 X
s ms
\contro,
ZN
4
E
3 2
n kU
1
N
I
0
0
i Sum of
/ B
& •
1'o all
2'0
preceding EPPs (V)
Fig. 5. Rundown of mean EPP amplitude at a normal (triangles)and a regenerating (crosses, 14 days after nerve crush) neuromuscular junction of the extensor digitorum longus, stimulated at ]0 Hz in the presence of hemicholinium-3 (4 pM). The abscissa shows the sum of all preceding EPPs, in volts. Qualitatively similar results were obtained from 4 other matched reinnervating and control muscle fibers.
/
Control
f
10 m s
Fig. 4. A Typical EPPs recorded f r o m a reinnervating extensor digitorum longus muscle fiber, before and after 2,3-butanedione monoxime (BDM, 20 mM). The muscle was denervated 15 days previously. Resting membrane potential was approximately - 30 mV, and each trace shows the average of 20 individual recordings. B Example of multiple innervation being "uncovered" by BDM. The two EPC recordings are from an endplate of extensor digitorum longus muscle denervated 19 days previously. Stimulus parameters were identical before and after BDM (20 raM), but only one EPC was seen in response to stimulation before BDM, even though high intensity stimulus was used. (Holding potential --65 mV, each trace is the average of 20 recordings)
causing asynchronous release of transmitter. Such alterations obviously make estimates of z very dubious, and it was found that most EPCs/EPPs produced in the presence of BDM could not be fitted to simple exponentials. Best-fit values from 35 EPC recordings (12 muscles, 1 2 - 1 9 days after nerve crush) were 4.16 -t- 0.23 ms (control) and 16.9 _+ 2.2 ms (with 20 m M BDM). The z values of MEPCs and MEPPs were also increased by BDM.
EfJects of B D M on "'quiescent" nerve terminals In addition to increasing the amplitude of EPCs at the reinnervating endplate, in some cases B D M enabled a neurally evoked EPC to be produced at an endplate where there was previously no activity. This activation of quiescent endplates, comparable to the action of 3,4-DAP, was seen with 20 m M BDM especially at the early stages of reinnervation ( 1 0 - 1 6 days). BDM was also able to "uncover" multiple innervation at the regenerating endplate. At many endplates which appeared singly innervated, treatment with BDM revealed two or more innervations. An example of this effect is shown in Fig. 4b. Variation of the stimulation
parameters often enabled the component EPCs to be separated, as described for regenerating NMJs in the absence of BDM [I 5]. B D M (20 raM) uncovered multiple innervations in this manner 1 3 - 2 6 days after nerve crush. The phenomenon was not noted with 3,4-DAP. The complex EPCs as illustrated in Fig. 4b are not likely to result from repetitive firing of the nerve terminal, since it was noted that at some endplates the first peak would always be larger than the subsequent peak/s, while at other endplates it would always be smaller.
Spontaneous activity due to B D M A further action of BDM was apparent which was confined to the period 2 0 - 24 days after nerve crush. Application of BDM (20 raM) caused bursts of spontaneous EPC activity, at approximately 1 - 2 Hz. The frequency of the bursts gradually declined, until they had disappeared after 15 rain of contact with BDM. This effect of BDM was highly reproducible, and was only seen during this narrow time band of reinnervation.
Effect of hemicholinium-3 While the response to temperature, [Ca2+]o, 3,4-DAP, and BDM indicated that regenerating nerve terminals have a reserve of quanta enabling them to respond to appropriate stimuli with an increase of m, the reserve is less than normal. This was apparent for preparations continuously stimulated in the presence of hemicholinium-3. Stimulation of reinnervating preparations caused rapid cessation of quantal release when hemicholinium-3 was used (Fig. 5).
EPC decay during reinnervation As Fig. 1 shows, EPCs decay slowly at the beginning of functional reinnervation of muscle. Specifically, the time
260 Table 1. Effect ofneostigmine (1.0 gM) on the time constant of EPC decay in the rat extensor digitorum longus Days after nerve crush
Time constant (ms)
Normal 11 - 14
Control
Neostigmine
1.46 _+ 0.04 4.47 _+ 0.70
13.50 _+ 0.98 9.99 _+ 0.51
Data are shown as mean • SEM, 10 fibers (at least 8 muscles) for each point. Holding potential - 6 0 mV
nA 1501
loo4 &
[] 5o-l -60 -50 L~ I
-40 I
A A
[]
[]
-30 I
A []
-20 I z~ L~
- 10 I
[]
A
A
J
Z~ =
i
=
10
20
30
40
5'0 mV
-50 -100
-150
Fig. 6. Typical current/voltage relationship for the EPC amplitude of a normal (squares) and a reinnervating (triangles) endplate
constant of EPC decay (z) was 6.00 _+ 0.69 ms (12 recordings from 6 muscles) at 12 days after nerve crush while the control value was 1.51 _+ 0.02 ms (156 recordings from 41 muscles). It is unlikely that this prolongation can be explained in terms of a denervation-induced reduction of endplate cholinesterase activity, since exposure to neostigmine (i.0 gM, 5 min) significantly prolonged ~ in both control and reinnervating preparations (Table 1). In addition to this prolonged monoexponential decay, it was also found that 30% of the EPCs examined between 11 and 22 days after nerve crush followed a double exponential decay. The mean values of T for the rapid and slow phases of decay were 1.70___ 0.26 and 3.51 _+ 0.19 ms, respectively. After 22 days such complex EPC decay was no longer detected, and z gradually recovered to normal by 30 days after nerve crush. It is interesting to note that while none of the hundreds of control EPCs examined at 22~ followed a double exponential decay, they occasionally did so when the temperature was lowered to 12~C. It has been reported [8] that two Lorentzian curves are sometimes necessary to describe the power spectrum of acetylcholine-evoked noise at the innervated endplate of frog muscle at 8 11~ Although EPCs were abnormal in amplitude and time course of decay early during reinnervation they continued to exhibit normal voltage sensitivity (Fig. 6). That is, amplitude remained a linear function of the holding potential and the average reversal potential was equivalent to control ( - 3 mV in both cases). Also,just as for normal
endplates, values of ~ were less at more positive holding potentials. However, ~ was more sensitive to voltage for reinnervating endplates; i.e. at reinnervating endplates there was an e-fold change in ~ for a 45 mV change of holding potential, while in controls a similar change of occurred with a 170 mV shift of holding potential.
Discussion The results presented here are in agreement with earlier reports of low quantal release of acetylcholine from regenerating motor nerve terminals in mouse [23] and frog [7]. As in the latter tissue, elevation of temperature or [Ca 2+]o increased m for regenerating NMJs. In addition, we studied the effects of drugs capable of enhancing transmission across the regenerating NMJ. The K § channel blocker 3,4-DAP increased m to normal during the early days of reinnervation, and EPPs could be elicited at early times after nerve crush. The phosphatase BDM also increased transmitter release and enabled EPPs/ EPCs to be recorded from quiescent endplates. The action of 3,4-DAP at very early times after nerve crush is particularly interesting in view of the "non-transmitting" stage of synapse formation found in some species during reinnervation of muscle. It has previously been reported [17] that MEPP frequency and amplitude were both normal before EPPs could be recorded at regenerating NMJs in adult frog muscle. In contrast to the amphibian muscle, a "non-transmitting" stage is at best infrequent during reinnervation of mammalian muscle [6, 23], and our own work has suggested that EPPs and MEPPs reappear at the same time during the reinnervation of rat hind-limb muscle [15]. Nevertheless, our present finding that EPPs/EPCs can be elicited at early times after nerve crush when preparations are pharmacologically treated justifies extension of the hypothesis of Dennis and Miledi [6] to regenerating mammalian NMJs. 3,4-DAP is known to block K § channels in pre-synaptic nerve membrane, thereby increasing the Ca 2 § influx into motor nerve terminals [14]. The ability of 3,4-DAP to activate transmitter release early in regeneration is therefore not surprising. Our data suggest that a "non-transmitting" phase of synapse formation may be overcome by increasing Ca 2 § influx. Although the exact mechanism by which BDM facilitates transmission has not been identified, it has been suggested [11] that BDM can dephosphorylate K + channels and inactivate them, hence prolonging the nerve terminal depolarisation in response to an action potential. It appears that BDM may have both pre- and postsynaptic effects, increasing transmitter release and prolonging the mean open time of the channels associated with acetylcholine receptors. A denervation-induced reduction in cholinesterase activity cannot fully explain the prolongation of EPCs and MEPCs that we observed during reinnervation, since neostigmine prolonged ~ at both normal and regenerating NMJs (Table 1), albeit to a slightly lesser extent at the latter. This observation is comparable to that made at the developing mammalian NMJ [10]. A second analogy with
261 the d e v e l o p i n g N M J is the o c c u r r e n c e o f c o m p l e x E P C d e c a y d u r i n g r e i n n e r v a t i o n o f a d u l t r a t h i n d - l i m b muscle. These findings c a n be i n t e r p r e t e d as a d e n e r v a t i o n i n d u c e d a l t e r a t i o n o f the o p e n time o f e n d p l a t e c h a n n e l s a s s o c i a t e d with the a c e t y l c h o l i n e receptor. A l t h o u g h we f o u n d t h a t E P P a n d M E P C d e c a y was p r o l o n g e d d u r i n g r e i n n e r v a t i o n , o t h e r w o r k e r s [3] h a v e f o u n d t h a t M E P P s a n d E P P s follow a n o r m a l time c o u r s e at r e g e n e r a t i n g N M J s in r a t d i a p h r a g m . B r e n n e r a n d S a k m a n n [4] r e p o r t e d t h a t the c h a n n e l o p e n time is n o t c h a n g e d d u r i n g r e i n n e r v a t i o n . This c o n c l u s i o n was b a s e d on three successful e x p e r i m e n t s in w h i c h they a n a l y z e d the s p e c t r u m o f a c e t y l c h o l i n e - i n d u c e d e n d p l a t e noise 1 8 - 20 d a y s after d e n e r v a t i n g the r a t d i a p h r a g m . T h e r e are c o n s i d e r a b l e differences in t e c h n i q u e b e t w e e n their s t u d y a n d o u r o w n , in p a r t i c u l a r the fact t h a t we a l l o w e d the nerve t e r m i n a l itself to " a p p l y " the acetylcholine, w h i c h m i g h t be o f significance if there are several dist a n t l y l o c a t e d release sites o n the r e g e n e r a t i n g nerve term i n a l [5]. I n a d d i t i o n , a differential effect o f p e r h y d r o h i s t r i o n i c o t o x i n on E P C s p r o d u c e d b y i o n t o p h o r e t i c a l l y versus n e u r a l t y a p p l i e d a c e t y l c h o l i n e has been n o t e d [1]. T h e r e f o r e , it is p o s s i b l e t h a t differences in m e t h o d o f a p p l i c a t i o n o f a c e t y l c h o l i n e m a y c o n t r i b u t e to the difference b e t w e e n o u r f i n d i n g a n d t h a t o f B r e n n e r a n d S a k m a n n [4]. O u r o w n w o r k has c o n s i s t e n t l y s h o w n p r o l o n g a t i o n o f M E P P a n d E P P d e c a y t i m e in r a t h i n d - l i m b muscles (e. g. [16]), c o n s i s t e n t w i t h a n i n c r e a s e d c h a n n e l o p e n time. P r o l o n g a t i o n o f M E P P d e c a y has also been r e p o r t e d at the r e i n n e r v a t i n g frog N M J [17].
Acknowledgements. We thank Dr. Stanley Von Hagen for his help in the statistical and graphical presentation of the data. This work was supported by grants NS-11055-09 from the National Institute of Neurological Disorders and Stroke, and 5 R01 AA08025 from the National Institute on Alcohol Abuse and Alcoholism. References
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