Br. J. Pharmacol. (1990), 99, 721-726
(D Macmillan Press Ltd, 1990
The effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles S.S. Kelly, 1C.B. Ferry & J.P. Bamforth Pharmacological Laboratories, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET 1 The purpose of this investigation was to determine the long-term effects of a single dose of persistent anticholinesterases on muscle action potentials evoked by nerve stimulation. 2 Action potentials (APs), elicited by stimulation of the phrenic nerve, were recorded intracellularly in muscle fibres of mouse diaphragm. The time between stimulus and AP was measured and the variability of this latency was calculated during trains of APs. At the beginning of trains of APs there was an increase in latency, and this delay was also measured. 3 Within 3h of subcutaneous injection, a single dose (500nmolkg-1) of the anticholinesterase, ecothiopate produced about 90% reduction in the acetylcholinesterase activity of homogenates of mouse diaphragm muscle, but five days after injection, this activity was not different from values in untreated animals. The initial delay of APs and the variability of latencies were increased four fold and two fold respectively, remained at these maxima from the 1st to the 5th day after ecothiopate, and returned to the values in untreated animals between 15 and 27 days after ecothiopate. 4 These effects of ecothiopate on AP latency were dose-dependent and were also seen in extensor digitorum longus and soleus muscles. 5 Other anticholinesterases used were BOS (pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate), a quaternary compound, and diisopropyl fluorophosphate, a tertiary compound, which had effects similar to those of ecothiopate; the greater duration of the effects of this compound may be related to the greater duration of reduction in cholinesterase activity. 6 Ecothiopate had no effect on the delay or variability of latencies of endplate potentials which were recorded in cut-fibre preparations 5 days later. 7 It is concluded that the effects of ecothiopate on the latencies of indirectly-evoked muscle APs are postjunctional, may not be related to the degree of reduction in cholinesterase activity at the time of recording, and are not directly linked to necrosis.
Introduction Subcutaneous injection of the organophosphate anticholinesterase, ecothiopate, causes necrosis at the endplate of mouse
diaphragm (Ferry & Townsend, 1986) which reaches a maximum 6-12h after injection. However, only a minority of the muscle fibres show necrosis and the question arises as to whether those fibres without structutal damage are more subtly affected. Transmission of excitation from nerve to the muscle and propagation of the action potential along the muscle might be compromised in damaged fibres. The clinical technique of single fibre electromyography (SFEMG) involves recording extracellularly the current field due to action potentials in single muscle fibres and may be used to measure jitter, the variability in the latency of a series of action potentials (APs). This variability may be in the time between APs of two fibres of the same motor unit in voluntary activity (Stalberg & Trontelj, 1979), or in the time between stimuli and APs in a fibre during a train of evoked responses (Trontelj et al., 1986). Excessive jitter is found in patients with muscular dystrophy or with myasthenia gravis, or after drugs (e.g. (+ )-tubocurarine), and is a sensitive indicator of muscular or neuromuscular disease. Jitter was considered by Stalberg & Trontelj (1979) to originate in the interaction of the endplate potential (e.p.p.) and the threshold of the muscle membrane during transmission. In the experiments in vitro described here, a new method of measuring jitter was developed. Action potentials evoked by nerve stimulation were recorded intracellularly which ensured that successive responses would be recorded from the same part of a single fibre. In order that all sources of jitter, presynaptic or postsynaptic, would be included, records were made 1
Author for correspondence.
close to the end of the muscle fibre near the tendon, but in some experiments records were made at the endplate. The membrane potential and overshoot of the AP are sensitive indicators of damage and provided criteria for excluding from further analysis any fibres damaged by the microelectrode during the contraction. The effect of anticholinesterases (antiChEs) on jitter was studied using several antiChEs to exclude the possibility that effects were other than a consequence of inhibition of acetylcholinesterase (AChE). Attempts were made to assess the role of AChE inhibition in the manifestation of jitter.
Methods Male albino mice aged 6-7 months were used in all experiments. At various times after subcutaneous injection of drugs, animals were killed by decapitation. The left hemidiaphragm and phrenic nerve were removed, pinned to Sylgard in a Perspex bath and irrigated with a physiological saline of the following composition (mM): NaCl 137, NaHCO3 12, NaH2PO4 1, KCI 5, CaCl2 2, MgCl2 1, glucose 25 and gassed with 5% CO2 in 02 and maintained at 37.0 + 0.50C. The phrenic nerve was stimulated via a suction electrode with supramaximal pulses of 0.05 ms duration. To minimize muscle movement many pins were used to fix the preparation to the Sylgard and, if necessary, some muscle fibres were cut. For experiments on extensor digitorum longus (EDL) or soleus (SOL) muscles, mice were anaesthetized with halothane in 50% nitrous oxide in oxygen; the limb muscles and their nerve supply were then removed and recordings were made as with the diaphragm. Glass capillary intracellular microelectrodes filled with 3M KCI and of resistance 10-15 MC were used to record resting
722
S.S. KELLY et al.
membrane potentials (RMPs) and action potentials (APs) of uncut muscle fibres near the tendon. Endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) were recorded in cut fibres by an electrode inserted focally at the endplate where the risetime of the potentials was less than 1.1msmV-'. APs were displayed on an oscilloscope and recorded on an FM tape recorder (Racal Store 4) with a tape speed of 30 inches per second (i.p.s.). These records were then replayed at 15/16 i.p.s. and analysed by a PDP 11/03 minicomputer. The sampling rate of the analogue-to-digital converter was 20kHz and with a record/replay ratio of 32, the effective sampling rate of the system was 640 kHz. Trains of 30 APs were recorded from each muscle fibre and approximately 10 fibres were sampled from each muscle. The stimulation frequency was usually 30Hz and if recording conditions were stable also 10Hz in the same fibre. Occasionally, stimulation was at 1 Hz. Data from a fibre were rejected if, during the train, there was a decrease in the RMP of more than 5 mV or in the AP amplitude of more than 10%. In experiments with ecothiopate iodide in vitro, the preparation was exposed to a 0.5 gM solution for 30-40 min, then to physiological saline for 30 min before the recording was commenced. Unless the results with stimulation at 10 Hz and 30 Hz were different, only data for 30 Hz are presented.
Analysis of records Computer programs were devised to measure the amplitude, time course, and latency of each AP. The latency was the interval between the stimulus and a point on the rising phase at 10% of peak amplitude. These data were used to calculate the mean consecutive difference (MCD) of latencies of APs 11-30 (plateau), and the individual latencies of the first 16 APs with respect to the first AP of the train. The formula used to calculate the MCD was:
assium acetate. When dissolved and diluted in normal saline, the potassium injected was negligible (approximately 6 gmol kg- 1). Solutions of BOS and DFP were made up shortly before injection. All mice given anticholinesterases were also given atropine sulphate 0.7pmolkg-' at the same time. Some mice were given atropine only.
Statistical analysis Results are expressed as mean + 1 s.d. of values from 2 to 5 animals, with the number of muscle fibres in parentheses. The Kolmogorov-Smirnov non-parametric test was used and groups were taken to be significantly different if P < 0.05 (2tail). Where experimental values returned to normal a long time after injection, the one-tail test was used because the direction of the difference from untreated values was predictable.
Results
The electrophysiological characteristics of untreated mice In untreated animals the RMP recorded at the tendon end of the muscle fibres was -71 + 5 mV (42). The AP had a time to peak (from 10% peak) of 216 + 34ts (35) and an amplitude of 81 + 8 mV (35). These values were not affected by the frequency of stimulation or by administration of any of the antiChEs either in vivo or in vitro. Records of typical action potentials are shown in Figure 1.
The effect offrequency ofstimulation The effect on jitter of the stimulus frequency was investigated because there is a marked effect of frequency on the amplitude of e.p.ps, which then may affect the latencies of the APs elicited. In untreated preparations, with stimulation at 10 or
MCD={ILI, -L121+1L12-LL131+ +L29-L301}/19 Where Ln is the latency of AP number n. The use of the MCD as a measure of variability may reduce the effects of any long-term drifts in latency, but this technique might hide initial changes at the beginning of trains of APs (Ekstedt et al., 1974). In our experiments, the initial change is measured as the 'delay' of the sixteenth AP relative to the first AP (i.e. latency of AP 16 minus the latency AP 1). A pulse generator (Digitimer) was used to simulate action potentials at 10Hz, the value obtained for MCD was 2.4 Ys, which represents the intrinsic error of the process of record/ replay/analysis.
b
a 50
mV 0.5
ms
Measurement of AChE activity The activity of AChE was assayed in strips of the junctional region of hemidiaphragms. Each hemidiaphragm was cut about 2mm either side of the intramuscular nerve and the junctional strip of muscle weighted and homogenised in 5ml of phosphate buffer (0.1 M, pH 8.0). The homogenate was incubated for at least 30min with 5OMm ethopropazine, a selective inhibitor of butyrylcholinesterase (Bayliss & Todrick, 1953), sonicated and then centrifuged at 1500g for 15min at 4°C. The supernatant was assayed for cholinesterase activity by the method of Ellman et al. (1961). Cholinesterase activity was expressed as nmol acetylthiocholine hydrolysed per min per mg of wet muscle and is due mainly to the AChE activity of the endplate (Das, 1989). The anticholinesterases used were:- ecothiopate (S-(2-trimethylammoniumethyl)phosphorothioate iodide), pinacolyl
S-(2-trimethylaminoethyl)methylphosphonothioate (BOS), or diisopropyl fluorophosphate (DFP). Ecothiopate was made up from Phospholine Eyedrops (Ayerst). This comprised 12.5mg ecothiopate and 40mg pot-
Ca
a)
0l
6 8 4 AP number
12
Figure 1 Action potentials (APs) recorded at the tendon end of muscle fibres of diaphragm and elicited by stimulation of the nerve at 30 Hz. (a) Records of APs 1-5 from untreated mice, shown displaced vertically downwards from AP 1. (b) Five days after ecothiopate 0.5 pmol kg- . Note the increased latencies of APs relative to the first AP which is greater after ecothiopate. (c) Graph showing the mean latency of the first 10 APs relative to the first. (El) Indicate values from untreated mice (n = 42) and (-) represent values 5 days after ecothiopate (n = 40).
ANTICHOLINESTERASES AND JITTER OF MUSCLE IMPULSES
30 Hz the latency of successive APs increased progressively and reached a steady value by about the tenth AP. This increase is quantified as the delay of AP 16 relative to AP 1. The value of the delay at 10 Hz was 34 + 12 ps (10), which was not different from that at 30Hz, 32 + 22 ps (42). With stimulation at 1 Hz, the delay was 1 + 25 us (11), which was different from the value at 30 Hz. The MCD of the APs 11-30 at 10Hz was 9.7 + 4.6ps (44), and at 30 Hz, 9.9 + 3.6 .s (42). These values were not different from each other but were different from that at 1 Hz, 6.3 + 1.2 jus (10). In these untreated mice, the values of MCD and of delay were distributed unimodally (Figure 2a). It is concluded that in normal diaphragms there was progressive delay of APs at the beginning of a train elicited at 10 or 30 Hz, but not at 1 Hz. There was a non-progressive variation in latency of APs 11-30, the MCD, which was greater at the higher frequencies than at 1 Hz.
14
12
723
I
a
10:
8.
6 4:
36
2
46 z- 2
60 >60
gm
RMA-M 60
I
o1 0 Ca) 80
48
Bram 12 24
0
0
mm
F= 36
48
m
w 0
u0
4
0
lo d
The effects of antiChEs in necrotizing doses
L-
8-
.0
06-
mm
E4
2 0
Ecothiopate was given subcutaneously to a batch of mice and measurements of MCD and delay made at increasing intervals until the values were no longer different from those of untreated animals. Three hours after a dose of 0.5gmolkg-1 ecothiopate and atropine, which causes necrosis of some muscle fibres, the AChE activity of homogenates of the junctional region was approximately 10% of that of untreated animals (Table 1). One day after ecothiopate 0.5pmolkg-' the delay and MCD at 30Hz were increased to maximal values which persisted until day 5 (Table 2). At day 7 the delay and MCD showed smaller increase. Although the delay had returned to normal by 15 days, the MCD was increased, but had returned to normal by 27 days. In mice given only atropine the MCD and delay were never significantly different from normal. At 5 days after ecothiopate 0.5pmolkg-' the populations of MCD and delay were not unimodal; many values were greater than those in untreated preparations (Figures 2b and 3b), and a few fibres had very large values for MCD and delay. These fibres excepted, there was no correlation between MCD and delay because muscle fibres with large MCDs did not necessarily have large delays. About 5% of the fibres sampled were not analysed because the train lacked at least one AP. There was repetitive firing of APs in about 20% of fibres 5 days after ecothiopate 0.5 Iumol kg- '.
48
.M 60 >
48
60 >60
I 0o
12
24
36
MCD (ps) Figure 2 The distribution of values of the mean consecutive difference (MCD) of action potentials (APs) at 30Hz in (a) untreated muscle, and in muscle 5 days after (b) ecothiopate 0.5pmol kg-1, (c) pinacolyl S-2-trimethylaminoethyl)methylphosphonothioate (BOS) 8.0ymolkg-1, (d) BOS 1.0pmolkg-'. The arrows indicate the mean value of the population.
At 5 days the MCD and delay were still maximally increased, but the AChE was at the untreated normal value (Table 1). Other antiChEs given in necrotizing doses caused similar changes in AChE and in the delay and MCD of APs (Figure 2c and 3c; Table 3). After BOS 8 pmol kg-l or DFP 14pmol kg- , the AChE 3 h later was reduced to similar values found with ecothiopate 0.5 pmol kg- 1, but the reduction lasted longer (Table 1). Five days after BOS (8 ymol kg- ), the increased delay and MCD were not different from those after ecothiopate
Table 1 Acetylcholinesterase activity of homogenates of diaphragm muscle at various times after in vivo injection of ecothiopate (ECO), pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate (BOS) or diisopropyl fluorophosphate (DFP) Dose
(pmol kg-) Untreated ECO (0.5)
Time after injection
AChE activity (nmol min lmg
-
1.34 ± 0.43 (38) 0.17 + 0.04 (8)*'
3h
')
Percentage inhibition
0 87
**
ECO (0.5) ECO (0.3) ECO (0.1) BOS (8.0) BOS (8.0) BOS (8.0) BOS (1.0)
5 days 3h 3h 3h
5 days 15 days 3h
1.28 + 0.23 (11) 0.17 + 0.05 (8)*b 0.57 + 0.11 (8)*.b 0.18 + 0.05 (11)* 0.79 + 0.18 (4)* 1.16 + 0.19 (8)* 0.84 + 0.07 (4)*b
4 87 57 87 41 13
37
**
BOS (1.0)
5 days
0.96 + 0.11 (11)*
28
**
BOS (1.0) DFP (14) DFP (14) DFP (14) DFP (11) DFP (11)
l5days 3h
5days 14 days 3h
Sdays
60
1.22 + 0.19 (12) 0.12 + 0.02 (4)* 1.05 + 0.15 (5)* 1.30 + 0.09 (4) 0.28 + 0.05 (8)* 0.94 + 0.16 (7)*
9 91 22 3 79 30
Values are mean + s.d. of (n) observations. *Indicates significant difference from untreated mice. All values 3 h after drugs significantly different from each other except those marked (', b). **Between values indicates they are significantly different.
are
not
724
S.S. KELLY et al.
Table 2 Mouse phrenic nerve-diaphragm stimulated indirectly at 30 Hz, action potentials (APs) recorded at tendon end of muscle fibres Time after injection
Delay of AP16 (,s)
MCD (.us)
Untreated
32 + 22 (42)
9.9 + 3.6 (42)
Atropine ECO 1 day
32 + 19 (29) 103 + 36 (13)*
9.4 + 2.6 (29) 23.7 + 12.1 (13)*
3 days
107 + 50 (14)*
27.9 + 32.9 (13)*
5 days 7 days
119+ 104(40)* 61 ± 37 (18)*
25.6 + 28.0 (39)* 17.8 + 9.5 (18)*
15 days 27 days
48 ± 38 (24) 25 + 23 (34)
14.7 + 7.6 (24)* 10.1 + 3.4 (34)
Delay and the mean consecutive difference (MCD) of APs at various times after ecothiopate 0.55smol kg'- (ECO) are shown. Values are mean ± s.d. of (n) observations. Values marked * are significantly different from untreated mice; values either side of # are not different.
(0.5pmol kg-1). With DFP 14.umol kg-', at 5 days the delay was the same as after BOS or ecothiopate; the MCD was not different from that after BOS but greater than after ecothiopate. As with ecothiopate, after BOS and DFP the increase in MCD and delay outlasted the inhibition of AChE. After BOS the AChE was normal at 15 days, when the delay and MCD were still increased. The delay had returned to normal by day 35, and the MCD by day 60 (Table 2). After DFP, the AChE was normal by day 14 and the MCD and the delay, by day 27 (Table 3).
than that of untreated preparations, whilst the MCD of 11.8 + 5.4 ps (25) was not different. Thus, the acute effects of inhibition of AChE after ecothiopate in vitro are exerted to a greater extent on delay than on MCD and are much more frequency-dependent than the effects of ecothiopate 5 days after administration in vivo. It is concluded that antiChEs in necrotizing doses increased MCD and delay of trains of APs at 30Hz to a maximum value for 5 days, which thereafter recovered. The period when jitter was increased exceeded the period of recovery of AChE.
The effect of the acute administration ofecothiopate in
Microscopy ofendplates after ecothiopate (0.5 ymol kg-')
vitro The acute exposure of the phrenic nerve-diaphragm preparation to ecothiopate 0.5 gM in vitro caused 90% inhibition of AChE and increased the MCD and delay at 30 Hz to 15.7 + 6.3 (10) and 138 + 78 (10), values not different from those 5 days after ecothiopate (0.5 Mmol kg- 1) in vivo (Table 2). At 10Hz, the delay was 51 + 24 ps (17) which was greater
I
14 a
Table 3 The delay of the 16th action potential (30 Hz), with respect to the first action potential, and mean consecutive difference (MCD) of action potentials .11 to 30 after (a) necrotizing and (b) non-necrotizing doses of ecothiopate (ECO), pinacolyl S-2-trimethylaminoethyl)methylphosphonothioate (BOS) or diisopropyl fluorophosphate (DFP) injected in vivo
12. 10
86: 4: 2-36 U,
0 4-
a)
U, In
-0 0
0 :c 8
E
6: 4: 2:
(pmol kg 1) V~~~~A 144 180
MMFFMad M4?I IW m ..
mW
36
72
108
216
252
POA A
288 > 288
1
-36 0 -36
36 36
72 72
Days
a Necrotizing
after
dose
I
n -36 0
.0
z
108 144 180 216 252 288 >288
8: 6: 4
2.
Phase contrast microscopy of living diaphragms 24 h after a necrotizing dose of ecothiopate (0.5 ymol kg- 1) in vivo showed in some fibres that the endplate region either appeared swollen with hypercontracted subsynaptic sarcomeres or appeared granular and without cross-striations. Fibres with a granular endplate region or with junctional or extrajunctional contraction clots showed a weak local autofluorescence, when viewed by epifluorescence microscopy with excitation at 450-
108 144 10
180 216 252 288 > 288
injection
ECO (0.3) BOS (8.0) BOS (8.0)
5 5 15
BOS (8.0)
35
46 ± 20 (42)*
BOS (8.0) DFP (14) DFP (14)
60 5 14
37 + 18 (15) 153 ± 84 (14)* 52 ± 36 (20)*
DFP (14)
27
33 + 22 (24)
12.2 + 4.8 (24)
Delay (ps) 39 + 19 (44) 35 + 32 (29) 28 + 20 (21) 23 + 33 (18) 48 + 26 (21)*
MCD (ps) 10.8 + 5.6 (44) 17.0 + 7.9 (29)* 10.4 + 6.6 (21) 9.4 + 3.5 (18) 10.8 + 4.1 (21)
52 ± 34 (31)* 12.1 + 5.2 (31) 162 + 181 (59)* 53.3 ± 86.2 (59)* 45 ± 28 (28)* 12.0 + 3.8 (28)*
b Non-necrotizing dose
(ymol kg-1) 0
36
72
108 144
180
216
252
288
> 288
Delay G(s) Figure 3 The distribution of values of delay of action potential (AP) 16 relative to AP 1 in a train at 30Hz in (a) untreated muscle, or 5 days after (b) ecothiopate 0.5 pmol kg- 1, or (c) pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate (BOS) 8.O1umol kg-' and (d) BOS 1.0pmolkg- The arrows indicate the mean value of the population. .
MCD (us)
12.3 + 4.8 (42) 11.6 + 4.0 (14) 60.7 + 52.6 (14)* 16.6 + 11.6 (20)*
1
I
-36
Delay (}As)
ECO (0.1) BOS (1.0) BOS (1.0) BOS (1.0) DFP (11)
Days after
injection 5 5
15 30 5
Values are mean + s.d. of (n) observations. Values marked * significantly different from untreated mice; values either side of # are not different.
are
ANTICHOLINESTERASES AND JITTER OF MUSCLE IMPULSES Table 4 The delay of the 16th action potential (30 Hz), with respect to the first action potential, and the mean consecutive difference (MCD) of action potentials 11 to 30 in diaphragm, extensor digitorum longus (EDL) and soleus muscles five days after injection of 0.4 or 0.1pmolkg-1 ecothiopate (ECO) Muscle
Drug (pmol kg 1)
Diaphragmn EDL Soleus
Untreated ECO (0.4) ECO (0.1) Untreated ECO (0.4) ECO (0.1) Untreated ECO (0.4) ECO (0.1)
Delay (us)
MCD (,s)
32 ± 22 (42) 9.9 ± 3.6 (42) 71 77 (31)* 24.7 + 45.2 (31)* 39 + 18.8 (44) 10.8 ± 5.6 (44) 50 27 (32) 10.7 ± 2.9 (32) 78 45.7 (34)* 19 ± 22.3 (34)* 56 + 30.9 (28) 10.6 ± 6.2 (28) 40 + 22 (36) 9.2 + 3.3 (36) 110 ± 70.4 (30)* 26.3 + 20.1 (30)* 64 ± 32.4 (27)* 15.3 + 10.4 (27)*
Values are mean + s.d. of (n) observations. *Significantly different from untreated mice.
490 nm and with wavelengths > 525 nm filtered from the emitted beam. Resting membrane potentials and sometimes m.e.p.ps were recorded in undamaged fibres, but convincing RMPs were not recorded from autofluorescing parts of fibres or from endplates which had a granular appearance. It is concluded that such damaged fibres are electrically silent and hence inexcitable and would not have been included in the population showing excessive jitter.
The effect of non-necrotizing doses of antiChEs Experiments were done with lower doses of antiChEs. After ecothiopate 0.3 ymol kg-1 the necrosis was less than after ecothiopate 0.5 pmol kg - but greater than in untreated animals; thus for ecothiopate, 0.3pmolkg-1 is close to the threshold dose for necrosis, for O.lmolkg-1 did not cause necrosis (Das, 1989). The effects of ecothiopate 0.1 and 0.3pmolkg-1 on the delay, MCD and AChE, and the effects of the nonnecrotizing doser of BOS (1 pmol kg- 1) and DFP (11 mol kg ') are shown in Tables 1 and 3b and Figures 2c,d. Compared with untreated mice, 5 days after ecothiopate 0.3 pmolkg-1, only the delay was increased and to a lesser extent than after ecothiopate 0.5 umol kg-1 or BOS 8 pmol kg -1. After ecothiopate 0.1 ymol kg- ', the delay and MCD were not increased. Five days after BOS 1 pmol kg-1 the MCD was increased to a lesser extent than after BOS 8pmolkg-1, and the effect lasted up to 15 days. After DFP 11 umol kg-1 there was increased delay. It is concluded that increases in delay and MCD are not necessarily associated with necrosis, and that the extent and the duration of these increases are dose- and drug-dependent.
The effect ofecothiopate on limb muscles Experiments were done on limb muscles which are less necrotized than diaphragm by ecothiopate (Ferry, unpublished observations). The delay and MCD of diaphragm, extensor Table 5 The delay of the 16th e.p.p. (30Hz), with respect to the first e.p.p., and MCD of e.p.ps 11 to 30 five days after
ecothiopate (ECO), pinacolyl S42-trimethylaminoethyl) methylphosphonothiate (BOS) or diisopropyl fluorophosphate (DFP) injected in vivo Drug (pmol kg- 1) Untreated
ECO (0.5) BOS (8.0) DFP (14.0)
Delay (,s) 38 + 42 + 41 + 24 +
16 (17) 15 (24) 13 (14) 18 (15)
MCD
9.2 8.9 10.6 10.5
+ + + +
(.is)
3.2 (17) 3.2 (24) 3.2 (14) 5.0 (15)
There was no significant difference between values with ECO, BOS or DFP and those in muscles from untreated mice. Values are mean + s.d. of (n) observations.
725
digitorum longus (EDL) and, soleus (SOL) muscles were measured 5 days after ecothiopate 0.4 jmol kg -1, which causes necrosis of the diaphragm similar to ecothiopate 0.5 pmol kg-'. Untreated EDL and SOL had values for delay and MCD at 30Hz similar to diaphragm (Table 4), and after ecothiopate 0.4 pmol kg-' there was an increased MCD, similar in all three muscles, and an increased delay which was greater in SOL than in the others. After ecothiopate 0.1 pmolkg-1, there was increased delay and MCD in SOL but not in EDL or in diaphragm (Tables 3 and 4). Thus SOL was more sensitive to ECO than the other muscles.
Latencies ofe.p.ps in untreated and ecothiopate-treated diaphragms To investigate if the increased variability in latencies of APs was attributable to increased variability of synaptic events, experiments were done on muscle fibres cut to prevent APs. The electrode was inserted to record m.e.p.ps focally, and the latencies of e.p.ps evoked at 30 Hz were measured. The results were that the delay and MCD of the e.p.ps were not different from those of APs recorded at the tendon end of untreated muscle. It is concluded that in untreated mice the variability of latencies within the train of e.p.ps is sufficient to account for the variability of latencies of a train of APs recorded near the tendon, and conduction along the muscle fibre causes no further increase in variability. The MCD and delay of e.p.ps 5 days after ecothiopate 0.5 umol kg-1 were the same as in untreated preparations (Table 5). It is concluded that the increased delay and MCD after ecothiopate cannot be due to increased variation in the timing of release or action of the transmitter.
Discussion In these experiments, changes in the latency of APs in a train of 30 stimuli were measured by the delay which represents a systematic increase during the change from rest to a steady state of activity, and by the MCD representing a nonsystematic variability of latency at this steady state. That the delay and MCD of APs in untreated muscle is frequencydependent and attributable to the delay and MCD of e.p.ps indicates that this jitter, like the variable quantum content of the e.p.p. may be a normal aspect of neuromuscular transmission (Ferry & Kelly, 1988). After ecothiopate the MCD and delay of APs at the tendon is increased without increase in the delay and MCD of the e.p.p. Thus the increased jitter is not due to exacerbation of those factors responsible for normal jitter, but to the induction of another factor which appears to be postsynaptic. The mechanism and site of this factor is currently under investigation. This additional source of jitter after antiChEs results in the distribution of delay and MCD becoming multimodal. After ecothiopate or BOS the distributions contained values indicating that some fibres had apparently normal jitter, some fibres had been moderately affected and some severely affected. In addition to these fibres all of which had carried the train of 30 APs and so had been subject to analysis, there was a fourth population of fibres which was excitable but failed during the train and, thereby, was excluded from the analysis, and a fifth population of fibres which was necrotic and electrically silent. Thus the measurement of jitter underestimates the total damage to muscle. Regeneration of necrotized fibres begins 2 days after ecothiopate and at 3 days some myoblasts contain regenerated myofibrils (Townsend, 1988). Regenerating necrotized fibres become functionally innervated after 7 days (Grubb & Harris, 1986), and it may be that the few fibres with very large values of MCD and delay could be regenerating.
726
S.S. KELLY et al.
The extent to which the increased jitter is secondary to the necrosis produced by the antiChEs is questionable. Clearly necrotic cells do not contribute to jitter, for they are electrically silent; and 5 days after non-necrotizing doses of antiChE there is still increased delay and MCD. Thus necrosis is not essential for the appearance of increased jitter. So it may be that the increased jitter in the diaphragm 5 days after a necrotizing dose was in those fibres surviving. Although there is increased delay and MCD after antiChEs it is likely that these phenomena are not directly related. There is no correlation between the two parameters in the same muscle fibres, untreated or after ecothiopate, except in a few severely affected fibres. Ecothiopate in vitro, has a greater effect on delay than on MCD. Delay recovers before MCD after ecothiopate in vivo. Furthermore, low doses of BOS increased MCD but not delay, whereas low doses of ecothiopate increased delay more than MCD. Thus it seem that the increased MCD and delay have different causes. Whether or not the inhibition of AChE at the time of the experiment determines the degree of jitter depends on the interpretation of the biochemical assay of AChE activity of muscle homogenates. It has been found that the jitter after acute exposure to ecothiopate in vitro, when inhibition of muscle AChE was maximal, is not different from those peak values of MCD and delay 5 days after injection in vivo when AChE was not significantly different from values in untreated
diaphragm. This indicates that, although increased jitter is a consequence of AChE inhibition, it does not depend upon the degree of inhibition determined at the same time as the jitter. However, the AChE activity of muscle homogenates measured several days after administration of an antiChE may not represent only the activity of the functional AChE responsible for terminating transmitter action. In our experiments made 5 days after the administration of ecothiopate, about 20% of fibres exhibited repetitive firing after a single stimulus which indicates prolongation of transmitter action, due to inhibition of the functional AChE (Ferry, 1988). Thus at this time, the biochemical determination of AChE activity is not a measure only of the functional AChE responsible for terminating transmitter action; perhaps some other non-functional AChE is also measured. Whilst it is clear that the increased jitter is a consequence of the inhibition of AChE, the relation between these two remains unclear. The differences in jitter 5 days after ecothiopate 0.5 and 0.3 imol kg- 1, BOS 8 pmol kg-' and DFP 14pmolkg-1, all of which cause similar inhibition of AChE 3h after administration, indicate that the maximal extent of inhibition is not sufficient to account for the increase in jitter. Perhaps the rate of onset of inhibition and its persistence may be factors in the aetiglogy of increased jitter. The relation between AChE activity and jitter thus remains to be determined.
References BAYLISS, B.J. & TODRICK, A. (1953). The use of specific inhibitors in the estimation of pseudocholinesterase in nervous tissue. J. Biochem., 54, 29. DAS, S.K. (1989). Mechanisms of anticholinesterase-induced myopathy and its prevention. Ph.D. Thesis. Aston University. EKSTEDT, J., NILSSON, G. & STALBERG, E. (1974). Calculation of the electromyographic jitter. J. Neurol. Neurosurg. Psychiatry, 37, 526-539. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V. & FEATHERSTONE,
R.M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88-95. FERRY, C.B. (1988). The origin of the anticholinesterase-induced repetitive activity of the phrenic nerve-diaphragm preparation of the rat in vitro. Br. J. Pharmacol., 94, 169-179. FERRY, C.B. & KELLY, S.S. (1988). The nature of the presynaptic effects
of (+)-turbocurarine at the mouse neuromuscular junction. J. Physiol., 403, 425-437. FERRY, C.B. & TOWNSEND, H.E. (1986). Anticholinesterase myopathy and changes in creatine kinase in mice. J. Physiol., 373, 29P. GRUBB, B.D. & HARRIS, J.B. (1986). The development of neuromuscular junctions on regenerating skeletal muscle fibres. J. Physiol., 380, 65P. STALBERG, E. & TRONTELJ, J.V. (1979). Single Fibre Electromyography. pp. 93-130, Old Woking, Surrey: The Mirvalle Press. TOWNSEND, H.E. (1988). Toxic effects of anticholinesterases on muscle. Ph.D. Thesis. Aston University. TRONTELJ, J.V., MIHELIN, M., FERNANDEZ, J.M. & STALBERG, E.
(1986). Axonal stimulation for end-plate jitter studies. J. Neurol. Neurosurg. Psychiatry, 49, 677-685. (Received October 24, 1989 Accepted November 16, 1989)
(D Macmillan Press Ltd, 1990
The effects of anticholinesterases on the latencies of action potentials in mouse skeletal muscles S.S. Kelly, 1C.B. Ferry & J.P. Bamforth Pharmacological Laboratories, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET 1 The purpose of this investigation was to determine the long-term effects of a single dose of persistent anticholinesterases on muscle action potentials evoked by nerve stimulation. 2 Action potentials (APs), elicited by stimulation of the phrenic nerve, were recorded intracellularly in muscle fibres of mouse diaphragm. The time between stimulus and AP was measured and the variability of this latency was calculated during trains of APs. At the beginning of trains of APs there was an increase in latency, and this delay was also measured. 3 Within 3h of subcutaneous injection, a single dose (500nmolkg-1) of the anticholinesterase, ecothiopate produced about 90% reduction in the acetylcholinesterase activity of homogenates of mouse diaphragm muscle, but five days after injection, this activity was not different from values in untreated animals. The initial delay of APs and the variability of latencies were increased four fold and two fold respectively, remained at these maxima from the 1st to the 5th day after ecothiopate, and returned to the values in untreated animals between 15 and 27 days after ecothiopate. 4 These effects of ecothiopate on AP latency were dose-dependent and were also seen in extensor digitorum longus and soleus muscles. 5 Other anticholinesterases used were BOS (pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate), a quaternary compound, and diisopropyl fluorophosphate, a tertiary compound, which had effects similar to those of ecothiopate; the greater duration of the effects of this compound may be related to the greater duration of reduction in cholinesterase activity. 6 Ecothiopate had no effect on the delay or variability of latencies of endplate potentials which were recorded in cut-fibre preparations 5 days later. 7 It is concluded that the effects of ecothiopate on the latencies of indirectly-evoked muscle APs are postjunctional, may not be related to the degree of reduction in cholinesterase activity at the time of recording, and are not directly linked to necrosis.
Introduction Subcutaneous injection of the organophosphate anticholinesterase, ecothiopate, causes necrosis at the endplate of mouse
diaphragm (Ferry & Townsend, 1986) which reaches a maximum 6-12h after injection. However, only a minority of the muscle fibres show necrosis and the question arises as to whether those fibres without structutal damage are more subtly affected. Transmission of excitation from nerve to the muscle and propagation of the action potential along the muscle might be compromised in damaged fibres. The clinical technique of single fibre electromyography (SFEMG) involves recording extracellularly the current field due to action potentials in single muscle fibres and may be used to measure jitter, the variability in the latency of a series of action potentials (APs). This variability may be in the time between APs of two fibres of the same motor unit in voluntary activity (Stalberg & Trontelj, 1979), or in the time between stimuli and APs in a fibre during a train of evoked responses (Trontelj et al., 1986). Excessive jitter is found in patients with muscular dystrophy or with myasthenia gravis, or after drugs (e.g. (+ )-tubocurarine), and is a sensitive indicator of muscular or neuromuscular disease. Jitter was considered by Stalberg & Trontelj (1979) to originate in the interaction of the endplate potential (e.p.p.) and the threshold of the muscle membrane during transmission. In the experiments in vitro described here, a new method of measuring jitter was developed. Action potentials evoked by nerve stimulation were recorded intracellularly which ensured that successive responses would be recorded from the same part of a single fibre. In order that all sources of jitter, presynaptic or postsynaptic, would be included, records were made 1
Author for correspondence.
close to the end of the muscle fibre near the tendon, but in some experiments records were made at the endplate. The membrane potential and overshoot of the AP are sensitive indicators of damage and provided criteria for excluding from further analysis any fibres damaged by the microelectrode during the contraction. The effect of anticholinesterases (antiChEs) on jitter was studied using several antiChEs to exclude the possibility that effects were other than a consequence of inhibition of acetylcholinesterase (AChE). Attempts were made to assess the role of AChE inhibition in the manifestation of jitter.
Methods Male albino mice aged 6-7 months were used in all experiments. At various times after subcutaneous injection of drugs, animals were killed by decapitation. The left hemidiaphragm and phrenic nerve were removed, pinned to Sylgard in a Perspex bath and irrigated with a physiological saline of the following composition (mM): NaCl 137, NaHCO3 12, NaH2PO4 1, KCI 5, CaCl2 2, MgCl2 1, glucose 25 and gassed with 5% CO2 in 02 and maintained at 37.0 + 0.50C. The phrenic nerve was stimulated via a suction electrode with supramaximal pulses of 0.05 ms duration. To minimize muscle movement many pins were used to fix the preparation to the Sylgard and, if necessary, some muscle fibres were cut. For experiments on extensor digitorum longus (EDL) or soleus (SOL) muscles, mice were anaesthetized with halothane in 50% nitrous oxide in oxygen; the limb muscles and their nerve supply were then removed and recordings were made as with the diaphragm. Glass capillary intracellular microelectrodes filled with 3M KCI and of resistance 10-15 MC were used to record resting
722
S.S. KELLY et al.
membrane potentials (RMPs) and action potentials (APs) of uncut muscle fibres near the tendon. Endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) were recorded in cut fibres by an electrode inserted focally at the endplate where the risetime of the potentials was less than 1.1msmV-'. APs were displayed on an oscilloscope and recorded on an FM tape recorder (Racal Store 4) with a tape speed of 30 inches per second (i.p.s.). These records were then replayed at 15/16 i.p.s. and analysed by a PDP 11/03 minicomputer. The sampling rate of the analogue-to-digital converter was 20kHz and with a record/replay ratio of 32, the effective sampling rate of the system was 640 kHz. Trains of 30 APs were recorded from each muscle fibre and approximately 10 fibres were sampled from each muscle. The stimulation frequency was usually 30Hz and if recording conditions were stable also 10Hz in the same fibre. Occasionally, stimulation was at 1 Hz. Data from a fibre were rejected if, during the train, there was a decrease in the RMP of more than 5 mV or in the AP amplitude of more than 10%. In experiments with ecothiopate iodide in vitro, the preparation was exposed to a 0.5 gM solution for 30-40 min, then to physiological saline for 30 min before the recording was commenced. Unless the results with stimulation at 10 Hz and 30 Hz were different, only data for 30 Hz are presented.
Analysis of records Computer programs were devised to measure the amplitude, time course, and latency of each AP. The latency was the interval between the stimulus and a point on the rising phase at 10% of peak amplitude. These data were used to calculate the mean consecutive difference (MCD) of latencies of APs 11-30 (plateau), and the individual latencies of the first 16 APs with respect to the first AP of the train. The formula used to calculate the MCD was:
assium acetate. When dissolved and diluted in normal saline, the potassium injected was negligible (approximately 6 gmol kg- 1). Solutions of BOS and DFP were made up shortly before injection. All mice given anticholinesterases were also given atropine sulphate 0.7pmolkg-' at the same time. Some mice were given atropine only.
Statistical analysis Results are expressed as mean + 1 s.d. of values from 2 to 5 animals, with the number of muscle fibres in parentheses. The Kolmogorov-Smirnov non-parametric test was used and groups were taken to be significantly different if P < 0.05 (2tail). Where experimental values returned to normal a long time after injection, the one-tail test was used because the direction of the difference from untreated values was predictable.
Results
The electrophysiological characteristics of untreated mice In untreated animals the RMP recorded at the tendon end of the muscle fibres was -71 + 5 mV (42). The AP had a time to peak (from 10% peak) of 216 + 34ts (35) and an amplitude of 81 + 8 mV (35). These values were not affected by the frequency of stimulation or by administration of any of the antiChEs either in vivo or in vitro. Records of typical action potentials are shown in Figure 1.
The effect offrequency ofstimulation The effect on jitter of the stimulus frequency was investigated because there is a marked effect of frequency on the amplitude of e.p.ps, which then may affect the latencies of the APs elicited. In untreated preparations, with stimulation at 10 or
MCD={ILI, -L121+1L12-LL131+ +L29-L301}/19 Where Ln is the latency of AP number n. The use of the MCD as a measure of variability may reduce the effects of any long-term drifts in latency, but this technique might hide initial changes at the beginning of trains of APs (Ekstedt et al., 1974). In our experiments, the initial change is measured as the 'delay' of the sixteenth AP relative to the first AP (i.e. latency of AP 16 minus the latency AP 1). A pulse generator (Digitimer) was used to simulate action potentials at 10Hz, the value obtained for MCD was 2.4 Ys, which represents the intrinsic error of the process of record/ replay/analysis.
b
a 50
mV 0.5
ms
Measurement of AChE activity The activity of AChE was assayed in strips of the junctional region of hemidiaphragms. Each hemidiaphragm was cut about 2mm either side of the intramuscular nerve and the junctional strip of muscle weighted and homogenised in 5ml of phosphate buffer (0.1 M, pH 8.0). The homogenate was incubated for at least 30min with 5OMm ethopropazine, a selective inhibitor of butyrylcholinesterase (Bayliss & Todrick, 1953), sonicated and then centrifuged at 1500g for 15min at 4°C. The supernatant was assayed for cholinesterase activity by the method of Ellman et al. (1961). Cholinesterase activity was expressed as nmol acetylthiocholine hydrolysed per min per mg of wet muscle and is due mainly to the AChE activity of the endplate (Das, 1989). The anticholinesterases used were:- ecothiopate (S-(2-trimethylammoniumethyl)phosphorothioate iodide), pinacolyl
S-(2-trimethylaminoethyl)methylphosphonothioate (BOS), or diisopropyl fluorophosphate (DFP). Ecothiopate was made up from Phospholine Eyedrops (Ayerst). This comprised 12.5mg ecothiopate and 40mg pot-
Ca
a)
0l
6 8 4 AP number
12
Figure 1 Action potentials (APs) recorded at the tendon end of muscle fibres of diaphragm and elicited by stimulation of the nerve at 30 Hz. (a) Records of APs 1-5 from untreated mice, shown displaced vertically downwards from AP 1. (b) Five days after ecothiopate 0.5 pmol kg- . Note the increased latencies of APs relative to the first AP which is greater after ecothiopate. (c) Graph showing the mean latency of the first 10 APs relative to the first. (El) Indicate values from untreated mice (n = 42) and (-) represent values 5 days after ecothiopate (n = 40).
ANTICHOLINESTERASES AND JITTER OF MUSCLE IMPULSES
30 Hz the latency of successive APs increased progressively and reached a steady value by about the tenth AP. This increase is quantified as the delay of AP 16 relative to AP 1. The value of the delay at 10 Hz was 34 + 12 ps (10), which was not different from that at 30Hz, 32 + 22 ps (42). With stimulation at 1 Hz, the delay was 1 + 25 us (11), which was different from the value at 30 Hz. The MCD of the APs 11-30 at 10Hz was 9.7 + 4.6ps (44), and at 30 Hz, 9.9 + 3.6 .s (42). These values were not different from each other but were different from that at 1 Hz, 6.3 + 1.2 jus (10). In these untreated mice, the values of MCD and of delay were distributed unimodally (Figure 2a). It is concluded that in normal diaphragms there was progressive delay of APs at the beginning of a train elicited at 10 or 30 Hz, but not at 1 Hz. There was a non-progressive variation in latency of APs 11-30, the MCD, which was greater at the higher frequencies than at 1 Hz.
14
12
723
I
a
10:
8.
6 4:
36
2
46 z- 2
60 >60
gm
RMA-M 60
I
o1 0 Ca) 80
48
Bram 12 24
0
0
mm
F= 36
48
m
w 0
u0
4
0
lo d
The effects of antiChEs in necrotizing doses
L-
8-
.0
06-
mm
E4
2 0
Ecothiopate was given subcutaneously to a batch of mice and measurements of MCD and delay made at increasing intervals until the values were no longer different from those of untreated animals. Three hours after a dose of 0.5gmolkg-1 ecothiopate and atropine, which causes necrosis of some muscle fibres, the AChE activity of homogenates of the junctional region was approximately 10% of that of untreated animals (Table 1). One day after ecothiopate 0.5pmolkg-' the delay and MCD at 30Hz were increased to maximal values which persisted until day 5 (Table 2). At day 7 the delay and MCD showed smaller increase. Although the delay had returned to normal by 15 days, the MCD was increased, but had returned to normal by 27 days. In mice given only atropine the MCD and delay were never significantly different from normal. At 5 days after ecothiopate 0.5pmolkg-' the populations of MCD and delay were not unimodal; many values were greater than those in untreated preparations (Figures 2b and 3b), and a few fibres had very large values for MCD and delay. These fibres excepted, there was no correlation between MCD and delay because muscle fibres with large MCDs did not necessarily have large delays. About 5% of the fibres sampled were not analysed because the train lacked at least one AP. There was repetitive firing of APs in about 20% of fibres 5 days after ecothiopate 0.5 Iumol kg- '.
48
.M 60 >
48
60 >60
I 0o
12
24
36
MCD (ps) Figure 2 The distribution of values of the mean consecutive difference (MCD) of action potentials (APs) at 30Hz in (a) untreated muscle, and in muscle 5 days after (b) ecothiopate 0.5pmol kg-1, (c) pinacolyl S-2-trimethylaminoethyl)methylphosphonothioate (BOS) 8.0ymolkg-1, (d) BOS 1.0pmolkg-'. The arrows indicate the mean value of the population.
At 5 days the MCD and delay were still maximally increased, but the AChE was at the untreated normal value (Table 1). Other antiChEs given in necrotizing doses caused similar changes in AChE and in the delay and MCD of APs (Figure 2c and 3c; Table 3). After BOS 8 pmol kg-l or DFP 14pmol kg- , the AChE 3 h later was reduced to similar values found with ecothiopate 0.5 pmol kg- 1, but the reduction lasted longer (Table 1). Five days after BOS (8 ymol kg- ), the increased delay and MCD were not different from those after ecothiopate
Table 1 Acetylcholinesterase activity of homogenates of diaphragm muscle at various times after in vivo injection of ecothiopate (ECO), pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate (BOS) or diisopropyl fluorophosphate (DFP) Dose
(pmol kg-) Untreated ECO (0.5)
Time after injection
AChE activity (nmol min lmg
-
1.34 ± 0.43 (38) 0.17 + 0.04 (8)*'
3h
')
Percentage inhibition
0 87
**
ECO (0.5) ECO (0.3) ECO (0.1) BOS (8.0) BOS (8.0) BOS (8.0) BOS (1.0)
5 days 3h 3h 3h
5 days 15 days 3h
1.28 + 0.23 (11) 0.17 + 0.05 (8)*b 0.57 + 0.11 (8)*.b 0.18 + 0.05 (11)* 0.79 + 0.18 (4)* 1.16 + 0.19 (8)* 0.84 + 0.07 (4)*b
4 87 57 87 41 13
37
**
BOS (1.0)
5 days
0.96 + 0.11 (11)*
28
**
BOS (1.0) DFP (14) DFP (14) DFP (14) DFP (11) DFP (11)
l5days 3h
5days 14 days 3h
Sdays
60
1.22 + 0.19 (12) 0.12 + 0.02 (4)* 1.05 + 0.15 (5)* 1.30 + 0.09 (4) 0.28 + 0.05 (8)* 0.94 + 0.16 (7)*
9 91 22 3 79 30
Values are mean + s.d. of (n) observations. *Indicates significant difference from untreated mice. All values 3 h after drugs significantly different from each other except those marked (', b). **Between values indicates they are significantly different.
are
not
724
S.S. KELLY et al.
Table 2 Mouse phrenic nerve-diaphragm stimulated indirectly at 30 Hz, action potentials (APs) recorded at tendon end of muscle fibres Time after injection
Delay of AP16 (,s)
MCD (.us)
Untreated
32 + 22 (42)
9.9 + 3.6 (42)
Atropine ECO 1 day
32 + 19 (29) 103 + 36 (13)*
9.4 + 2.6 (29) 23.7 + 12.1 (13)*
3 days
107 + 50 (14)*
27.9 + 32.9 (13)*
5 days 7 days
119+ 104(40)* 61 ± 37 (18)*
25.6 + 28.0 (39)* 17.8 + 9.5 (18)*
15 days 27 days
48 ± 38 (24) 25 + 23 (34)
14.7 + 7.6 (24)* 10.1 + 3.4 (34)
Delay and the mean consecutive difference (MCD) of APs at various times after ecothiopate 0.55smol kg'- (ECO) are shown. Values are mean ± s.d. of (n) observations. Values marked * are significantly different from untreated mice; values either side of # are not different.
(0.5pmol kg-1). With DFP 14.umol kg-', at 5 days the delay was the same as after BOS or ecothiopate; the MCD was not different from that after BOS but greater than after ecothiopate. As with ecothiopate, after BOS and DFP the increase in MCD and delay outlasted the inhibition of AChE. After BOS the AChE was normal at 15 days, when the delay and MCD were still increased. The delay had returned to normal by day 35, and the MCD by day 60 (Table 2). After DFP, the AChE was normal by day 14 and the MCD and the delay, by day 27 (Table 3).
than that of untreated preparations, whilst the MCD of 11.8 + 5.4 ps (25) was not different. Thus, the acute effects of inhibition of AChE after ecothiopate in vitro are exerted to a greater extent on delay than on MCD and are much more frequency-dependent than the effects of ecothiopate 5 days after administration in vivo. It is concluded that antiChEs in necrotizing doses increased MCD and delay of trains of APs at 30Hz to a maximum value for 5 days, which thereafter recovered. The period when jitter was increased exceeded the period of recovery of AChE.
The effect of the acute administration ofecothiopate in
Microscopy ofendplates after ecothiopate (0.5 ymol kg-')
vitro The acute exposure of the phrenic nerve-diaphragm preparation to ecothiopate 0.5 gM in vitro caused 90% inhibition of AChE and increased the MCD and delay at 30 Hz to 15.7 + 6.3 (10) and 138 + 78 (10), values not different from those 5 days after ecothiopate (0.5 Mmol kg- 1) in vivo (Table 2). At 10Hz, the delay was 51 + 24 ps (17) which was greater
I
14 a
Table 3 The delay of the 16th action potential (30 Hz), with respect to the first action potential, and mean consecutive difference (MCD) of action potentials .11 to 30 after (a) necrotizing and (b) non-necrotizing doses of ecothiopate (ECO), pinacolyl S-2-trimethylaminoethyl)methylphosphonothioate (BOS) or diisopropyl fluorophosphate (DFP) injected in vivo
12. 10
86: 4: 2-36 U,
0 4-
a)
U, In
-0 0
0 :c 8
E
6: 4: 2:
(pmol kg 1) V~~~~A 144 180
MMFFMad M4?I IW m ..
mW
36
72
108
216
252
POA A
288 > 288
1
-36 0 -36
36 36
72 72
Days
a Necrotizing
after
dose
I
n -36 0
.0
z
108 144 180 216 252 288 >288
8: 6: 4
2.
Phase contrast microscopy of living diaphragms 24 h after a necrotizing dose of ecothiopate (0.5 ymol kg- 1) in vivo showed in some fibres that the endplate region either appeared swollen with hypercontracted subsynaptic sarcomeres or appeared granular and without cross-striations. Fibres with a granular endplate region or with junctional or extrajunctional contraction clots showed a weak local autofluorescence, when viewed by epifluorescence microscopy with excitation at 450-
108 144 10
180 216 252 288 > 288
injection
ECO (0.3) BOS (8.0) BOS (8.0)
5 5 15
BOS (8.0)
35
46 ± 20 (42)*
BOS (8.0) DFP (14) DFP (14)
60 5 14
37 + 18 (15) 153 ± 84 (14)* 52 ± 36 (20)*
DFP (14)
27
33 + 22 (24)
12.2 + 4.8 (24)
Delay (ps) 39 + 19 (44) 35 + 32 (29) 28 + 20 (21) 23 + 33 (18) 48 + 26 (21)*
MCD (ps) 10.8 + 5.6 (44) 17.0 + 7.9 (29)* 10.4 + 6.6 (21) 9.4 + 3.5 (18) 10.8 + 4.1 (21)
52 ± 34 (31)* 12.1 + 5.2 (31) 162 + 181 (59)* 53.3 ± 86.2 (59)* 45 ± 28 (28)* 12.0 + 3.8 (28)*
b Non-necrotizing dose
(ymol kg-1) 0
36
72
108 144
180
216
252
288
> 288
Delay G(s) Figure 3 The distribution of values of delay of action potential (AP) 16 relative to AP 1 in a train at 30Hz in (a) untreated muscle, or 5 days after (b) ecothiopate 0.5 pmol kg- 1, or (c) pinacolyl S-(2-trimethylaminoethyl)methylphosphonothioate (BOS) 8.O1umol kg-' and (d) BOS 1.0pmolkg- The arrows indicate the mean value of the population. .
MCD (us)
12.3 + 4.8 (42) 11.6 + 4.0 (14) 60.7 + 52.6 (14)* 16.6 + 11.6 (20)*
1
I
-36
Delay (}As)
ECO (0.1) BOS (1.0) BOS (1.0) BOS (1.0) DFP (11)
Days after
injection 5 5
15 30 5
Values are mean + s.d. of (n) observations. Values marked * significantly different from untreated mice; values either side of # are not different.
are
ANTICHOLINESTERASES AND JITTER OF MUSCLE IMPULSES Table 4 The delay of the 16th action potential (30 Hz), with respect to the first action potential, and the mean consecutive difference (MCD) of action potentials 11 to 30 in diaphragm, extensor digitorum longus (EDL) and soleus muscles five days after injection of 0.4 or 0.1pmolkg-1 ecothiopate (ECO) Muscle
Drug (pmol kg 1)
Diaphragmn EDL Soleus
Untreated ECO (0.4) ECO (0.1) Untreated ECO (0.4) ECO (0.1) Untreated ECO (0.4) ECO (0.1)
Delay (us)
MCD (,s)
32 ± 22 (42) 9.9 ± 3.6 (42) 71 77 (31)* 24.7 + 45.2 (31)* 39 + 18.8 (44) 10.8 ± 5.6 (44) 50 27 (32) 10.7 ± 2.9 (32) 78 45.7 (34)* 19 ± 22.3 (34)* 56 + 30.9 (28) 10.6 ± 6.2 (28) 40 + 22 (36) 9.2 + 3.3 (36) 110 ± 70.4 (30)* 26.3 + 20.1 (30)* 64 ± 32.4 (27)* 15.3 + 10.4 (27)*
Values are mean + s.d. of (n) observations. *Significantly different from untreated mice.
490 nm and with wavelengths > 525 nm filtered from the emitted beam. Resting membrane potentials and sometimes m.e.p.ps were recorded in undamaged fibres, but convincing RMPs were not recorded from autofluorescing parts of fibres or from endplates which had a granular appearance. It is concluded that such damaged fibres are electrically silent and hence inexcitable and would not have been included in the population showing excessive jitter.
The effect of non-necrotizing doses of antiChEs Experiments were done with lower doses of antiChEs. After ecothiopate 0.3 ymol kg-1 the necrosis was less than after ecothiopate 0.5 pmol kg - but greater than in untreated animals; thus for ecothiopate, 0.3pmolkg-1 is close to the threshold dose for necrosis, for O.lmolkg-1 did not cause necrosis (Das, 1989). The effects of ecothiopate 0.1 and 0.3pmolkg-1 on the delay, MCD and AChE, and the effects of the nonnecrotizing doser of BOS (1 pmol kg- 1) and DFP (11 mol kg ') are shown in Tables 1 and 3b and Figures 2c,d. Compared with untreated mice, 5 days after ecothiopate 0.3 pmolkg-1, only the delay was increased and to a lesser extent than after ecothiopate 0.5 umol kg-1 or BOS 8 pmol kg -1. After ecothiopate 0.1 ymol kg- ', the delay and MCD were not increased. Five days after BOS 1 pmol kg-1 the MCD was increased to a lesser extent than after BOS 8pmolkg-1, and the effect lasted up to 15 days. After DFP 11 umol kg-1 there was increased delay. It is concluded that increases in delay and MCD are not necessarily associated with necrosis, and that the extent and the duration of these increases are dose- and drug-dependent.
The effect ofecothiopate on limb muscles Experiments were done on limb muscles which are less necrotized than diaphragm by ecothiopate (Ferry, unpublished observations). The delay and MCD of diaphragm, extensor Table 5 The delay of the 16th e.p.p. (30Hz), with respect to the first e.p.p., and MCD of e.p.ps 11 to 30 five days after
ecothiopate (ECO), pinacolyl S42-trimethylaminoethyl) methylphosphonothiate (BOS) or diisopropyl fluorophosphate (DFP) injected in vivo Drug (pmol kg- 1) Untreated
ECO (0.5) BOS (8.0) DFP (14.0)
Delay (,s) 38 + 42 + 41 + 24 +
16 (17) 15 (24) 13 (14) 18 (15)
MCD
9.2 8.9 10.6 10.5
+ + + +
(.is)
3.2 (17) 3.2 (24) 3.2 (14) 5.0 (15)
There was no significant difference between values with ECO, BOS or DFP and those in muscles from untreated mice. Values are mean + s.d. of (n) observations.
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digitorum longus (EDL) and, soleus (SOL) muscles were measured 5 days after ecothiopate 0.4 jmol kg -1, which causes necrosis of the diaphragm similar to ecothiopate 0.5 pmol kg-'. Untreated EDL and SOL had values for delay and MCD at 30Hz similar to diaphragm (Table 4), and after ecothiopate 0.4 pmol kg-' there was an increased MCD, similar in all three muscles, and an increased delay which was greater in SOL than in the others. After ecothiopate 0.1 pmolkg-1, there was increased delay and MCD in SOL but not in EDL or in diaphragm (Tables 3 and 4). Thus SOL was more sensitive to ECO than the other muscles.
Latencies ofe.p.ps in untreated and ecothiopate-treated diaphragms To investigate if the increased variability in latencies of APs was attributable to increased variability of synaptic events, experiments were done on muscle fibres cut to prevent APs. The electrode was inserted to record m.e.p.ps focally, and the latencies of e.p.ps evoked at 30 Hz were measured. The results were that the delay and MCD of the e.p.ps were not different from those of APs recorded at the tendon end of untreated muscle. It is concluded that in untreated mice the variability of latencies within the train of e.p.ps is sufficient to account for the variability of latencies of a train of APs recorded near the tendon, and conduction along the muscle fibre causes no further increase in variability. The MCD and delay of e.p.ps 5 days after ecothiopate 0.5 umol kg-1 were the same as in untreated preparations (Table 5). It is concluded that the increased delay and MCD after ecothiopate cannot be due to increased variation in the timing of release or action of the transmitter.
Discussion In these experiments, changes in the latency of APs in a train of 30 stimuli were measured by the delay which represents a systematic increase during the change from rest to a steady state of activity, and by the MCD representing a nonsystematic variability of latency at this steady state. That the delay and MCD of APs in untreated muscle is frequencydependent and attributable to the delay and MCD of e.p.ps indicates that this jitter, like the variable quantum content of the e.p.p. may be a normal aspect of neuromuscular transmission (Ferry & Kelly, 1988). After ecothiopate the MCD and delay of APs at the tendon is increased without increase in the delay and MCD of the e.p.p. Thus the increased jitter is not due to exacerbation of those factors responsible for normal jitter, but to the induction of another factor which appears to be postsynaptic. The mechanism and site of this factor is currently under investigation. This additional source of jitter after antiChEs results in the distribution of delay and MCD becoming multimodal. After ecothiopate or BOS the distributions contained values indicating that some fibres had apparently normal jitter, some fibres had been moderately affected and some severely affected. In addition to these fibres all of which had carried the train of 30 APs and so had been subject to analysis, there was a fourth population of fibres which was excitable but failed during the train and, thereby, was excluded from the analysis, and a fifth population of fibres which was necrotic and electrically silent. Thus the measurement of jitter underestimates the total damage to muscle. Regeneration of necrotized fibres begins 2 days after ecothiopate and at 3 days some myoblasts contain regenerated myofibrils (Townsend, 1988). Regenerating necrotized fibres become functionally innervated after 7 days (Grubb & Harris, 1986), and it may be that the few fibres with very large values of MCD and delay could be regenerating.
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The extent to which the increased jitter is secondary to the necrosis produced by the antiChEs is questionable. Clearly necrotic cells do not contribute to jitter, for they are electrically silent; and 5 days after non-necrotizing doses of antiChE there is still increased delay and MCD. Thus necrosis is not essential for the appearance of increased jitter. So it may be that the increased jitter in the diaphragm 5 days after a necrotizing dose was in those fibres surviving. Although there is increased delay and MCD after antiChEs it is likely that these phenomena are not directly related. There is no correlation between the two parameters in the same muscle fibres, untreated or after ecothiopate, except in a few severely affected fibres. Ecothiopate in vitro, has a greater effect on delay than on MCD. Delay recovers before MCD after ecothiopate in vivo. Furthermore, low doses of BOS increased MCD but not delay, whereas low doses of ecothiopate increased delay more than MCD. Thus it seem that the increased MCD and delay have different causes. Whether or not the inhibition of AChE at the time of the experiment determines the degree of jitter depends on the interpretation of the biochemical assay of AChE activity of muscle homogenates. It has been found that the jitter after acute exposure to ecothiopate in vitro, when inhibition of muscle AChE was maximal, is not different from those peak values of MCD and delay 5 days after injection in vivo when AChE was not significantly different from values in untreated
diaphragm. This indicates that, although increased jitter is a consequence of AChE inhibition, it does not depend upon the degree of inhibition determined at the same time as the jitter. However, the AChE activity of muscle homogenates measured several days after administration of an antiChE may not represent only the activity of the functional AChE responsible for terminating transmitter action. In our experiments made 5 days after the administration of ecothiopate, about 20% of fibres exhibited repetitive firing after a single stimulus which indicates prolongation of transmitter action, due to inhibition of the functional AChE (Ferry, 1988). Thus at this time, the biochemical determination of AChE activity is not a measure only of the functional AChE responsible for terminating transmitter action; perhaps some other non-functional AChE is also measured. Whilst it is clear that the increased jitter is a consequence of the inhibition of AChE, the relation between these two remains unclear. The differences in jitter 5 days after ecothiopate 0.5 and 0.3 imol kg- 1, BOS 8 pmol kg-' and DFP 14pmolkg-1, all of which cause similar inhibition of AChE 3h after administration, indicate that the maximal extent of inhibition is not sufficient to account for the increase in jitter. Perhaps the rate of onset of inhibition and its persistence may be factors in the aetiglogy of increased jitter. The relation between AChE activity and jitter thus remains to be determined.
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R.M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88-95. FERRY, C.B. (1988). The origin of the anticholinesterase-induced repetitive activity of the phrenic nerve-diaphragm preparation of the rat in vitro. Br. J. Pharmacol., 94, 169-179. FERRY, C.B. & KELLY, S.S. (1988). The nature of the presynaptic effects
of (+)-turbocurarine at the mouse neuromuscular junction. J. Physiol., 403, 425-437. FERRY, C.B. & TOWNSEND, H.E. (1986). Anticholinesterase myopathy and changes in creatine kinase in mice. J. Physiol., 373, 29P. GRUBB, B.D. & HARRIS, J.B. (1986). The development of neuromuscular junctions on regenerating skeletal muscle fibres. J. Physiol., 380, 65P. STALBERG, E. & TRONTELJ, J.V. (1979). Single Fibre Electromyography. pp. 93-130, Old Woking, Surrey: The Mirvalle Press. TOWNSEND, H.E. (1988). Toxic effects of anticholinesterases on muscle. Ph.D. Thesis. Aston University. TRONTELJ, J.V., MIHELIN, M., FERNANDEZ, J.M. & STALBERG, E.
(1986). Axonal stimulation for end-plate jitter studies. J. Neurol. Neurosurg. Psychiatry, 49, 677-685. (Received October 24, 1989 Accepted November 16, 1989)
