Pflfigers Arch (1982) 394 :202 - 210 Pfliigers Archly European Joomal of Physiology @ Springer-Verlag 1982 Postsynaptic Currents in Different Types of Frog Muscle Fibre V. V. Fedorov, L. G. Magazanik, V. A. Snetkov, and A. L. Zefirov* I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Thorez pr., 44, Leningrad, K-223, USSR Abstract. Miniature end-plate currents (mepcs) were recorded from fast and slow muscle fibres in two muscles, m. cruralis and m. ileofibularis, under voltage clamp or extracellularly. Both fibre types showed an identical sensitivity to c~- bungarotoxin, and an exponential dependence of mepc decay time-constant upon membrane potential, but there were some important differences : i) the rise time and half-time of decay were approximately 3 times longer in slow junctions than in fast ones; ii) inhibition of AChE had a more prominent effect on mepcs recorded in fast fibres while preserving the signif- icant difference in mepc time course between fast and slow fibres; iii) treatment by c~-bungarotoxin had a greater effect on mepc time course in slow junctions even in the absence of anticholinesterase; iv) a more prominent variability of mepc time course was observed in slow junctions, especially after treatment by anticholinesterase drugs. Fluctuations of conductance of the postjunctional mem- brane induced by application of acetylcholine to different frog muscle fibres have been analysed. The autocorrelation function of these fluctuations was exponential and gave information about the mean life-time of single ionic channels and their conductance. There were two main classes of ionic channels : "fast" with mean life-time 2.9 ms and conductance 18.3pS in single-innervated fast muscle fibres of phasic bundle of m. ileofibularis, m. sartorius and m. cutaneous pectoris; and "slow" -8.3 ms and 12.5 pS correspondingly (t = 8-10~ holding potential - 80mV) in multi-innervated fibres of tonic bundle of m. ileofibularis. An intermediate type of singly-innervated muscle fibres was found in tonic bundles of m. cruralis and m. ileofibularis. The time course of mepcs and life-time of ionic channels recorded in these fibres were intermediate between typical fast and slow mepcs. Simultaneous analysis ofmepcs and ACh-induced current fluctuations suggests that the decay of mepcs in typical fast fibres is governed mainly by the closing of ionic channels while the decay of most mepcs in slow fibres is substantially slower than the rate of channel closing. Evidently some additional factors may prolong the effect of ACh-quanta in slow junctions. The relationship between the functional properties of different muscle fibres and rates of sequential steps of postjunctional activation is discussed. Key words: Fast and slow muscle fibres - Synaptic currents - Cholinester~se inhibitors - Potential dependency - Voltage clamp - ACh-induced noise - Frogs OtJ))rint requests to Dr. L. G. Magazanik at the above address * Present address: Department of Normal Physiology, Kazan Medical Institute, Kazan, USSR Introduction Amphibian slow (or tonic) muscle fibres are characterized by relatively slow graded contractions that depend upon the extent of membrane depolarization evoked by multiple junctional potentials. These functional properties are related to a number of special physiological mechanisms (Zhukov 1969) including peculiarities ofinnervation. Rather short frog slow fibres (6-12mm length) have 3-10 synaptic inputs (Nasledov and Fedorov 1965; Hess 1970). The nerve ter- minals are delicate and have a small number of "active zones"; postjunctional infoldings are absent (Page 1965). Intracellularly recorded induced synaptic potentials (Kuffler and Vaughan Williams 1953) and currents (Oomura and Tomita 1960) reveal a relatively slow time course in frog slow muscle fibres. This fact was confirmed by investigation of miniature end-plate currents (mepcs) recorded under voltage clamp conditions or extracellularly in slow fibres from tonic bundle of frog ileofibularis (Magazanik et al. 1979). In the present paper, combination of these techniques was used to avoid the influence of spatial and time summation of responses generated by several synaptic inputs and spatial non-uniformity of the membrane potential. Study of the rate-limiting processes responsible for the observed slowness of mepc time course was continued using another frog slow muscle - m. cruralis (Gilly 1975). Simultaneous analysis of mepc time course and fluctuations of synaptic current induced by applied acetylcholine (ACh) was used to show whether the slow decay of mepcs in slow fibres is due to longer channel life-time. Preliminary results have been published elsewhere (Magazanik et al. 1979; Fedorov et al. 1981). Methods Preparations. Experiments were performed on the neuromus- cular junctions of m. cruralis and m. ileofibularis of the frog Rana temporaria (winter frogs, kept at 6- 8 ~C). The ventral (anterior) head of m. triceps femoris (m. cruralis) was dissected with the stump ofn. ischiadicus. This thin bundle is roughly triangular in shape and consists of fibres about 5- 10ram length. Morphological examination and force measurements indicate that this muscle is relatively rich in typical slow fibres (Gilly 1975). Several experiments were done on fast fibres of re. sartorius and m. cutaneous pectoris. Solutions. Bathing solution, of the following composition (raM): NaC1 115, KC1 2.5, CaC12 1.8, was buffered by 0031-6768/82/0394/0202/$01.80
Postsynaptic Currents in Different Types of Frog Muscle Fibre
V. V. Fedorov, L. G. Magazanik, V. A. Snetkov, and A. L. Zefirov*
I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Thorez pr., 44, Leningrad, K-223, USSR
A b s t r a c t . Miniature end-plate currents (mepcs) were recorded from fast and slow muscle fibres in two muscles, m. cruralis and m. ileofibularis, under voltage clamp or extracellularly. Both fibre types showed an identical sensitivity to c~- bungarotoxin, and an exponential dependence of mepc decay time-constant upon membrane potential, but there were some important differences : i) the rise time and half-time of decay were approximately 3 times longer in slow junctions than in fast ones; ii) inhibition of AChE had a more prominent effect on mepcs recorded in fast fibres while preserving the signif- icant difference in mepc time course between fast and slow fibres; iii) treatment by c~-bungarotoxin had a greater effect on mepc time course in slow junctions even in the absence of anticholinesterase; iv) a more prominent variability of mepc time course was observed in slow junctions, especially after treatment by anticholinesterase drugs.
Fluctuations of conductance of the postjunctional mem- brane induced by application of acetylcholine to different frog muscle fibres have been analysed. The autocorrelation function of these fluctuations was exponential and gave information about the mean life-time of single ionic channels and their conductance. There were two main classes of ionic channels : "fast" with mean life-time 2.9 ms and conductance 18.3pS in single-innervated fast muscle fibres of phasic bundle of m. ileofibularis, m. sartorius and m. cutaneous pectoris; and "slow" - 8 . 3 ms and 12.5 pS correspondingly (t = 8-10~ holding potential - 80mV) in multi-innervated fibres of tonic bundle of m. ileofibularis. An intermediate type of singly-innervated muscle fibres was found in tonic bundles of m. cruralis and m. ileofibularis. The time course of mepcs and life-time of ionic channels recorded in these fibres were intermediate between typical fast and slow mepcs.
Simultaneous analysis ofmepcs and ACh-induced current fluctuations suggests that the decay of mepcs in typical fast fibres is governed mainly by the closing of ionic channels while the decay of most mepcs in slow fibres is substantially slower than the rate of channel closing. Evidently some additional factors may prolong the effect of ACh-quanta in slow junctions. The relationship between the functional properties of different muscle fibres and rates of sequential steps of postjunctional activation is discussed.
Key words: Fast and slow muscle fibres - Synaptic currents - Cholinester~se inhibitors - Potential dependency - Voltage clamp - ACh-induced noise - Frogs
OtJ))rint requests to Dr. L. G. Magazanik at the above address * Present address: Department of Normal Physiology, Kazan Medical
Institute, Kazan, USSR
I n t r o d u c t i o n
Amphibian slow (or tonic) muscle fibres are characterized by relatively slow graded contractions that depend upon the extent of membrane depolarization evoked by multiple junctional potentials. These functional properties are related to a number of special physiological mechanisms (Zhukov 1969) including peculiarities ofinnervation. Rather short frog slow fibres ( 6 - 1 2 m m length) have 3 - 1 0 synaptic inputs (Nasledov and Fedorov 1965; Hess 1970). The nerve ter- minals are delicate and have a small number of "active zones"; postjunctional infoldings are absent (Page 1965). Intracellularly recorded induced synaptic potentials (Kuffler and Vaughan Williams 1953) and currents (Oomura and Tomita 1960) reveal a relatively slow time course in frog slow muscle fibres. This fact was confirmed by investigation of miniature end-plate currents (mepcs) recorded under voltage clamp conditions or extracellularly in slow fibres from tonic bundle of frog ileofibularis (Magazanik et al. 1979). In the present paper, combination of these techniques was used to avoid the influence of spatial and time summation of responses generated by several synaptic inputs and spatial non-uniformity of the membrane potential.
Study of the rate-limiting processes responsible for the observed slowness of mepc time course was continued using another frog slow muscle - m. cruralis (Gilly 1975). Simultaneous analysis of mepc time course and fluctuations of synaptic current induced by applied acetylcholine (ACh) was used to show whether the slow decay of mepcs in slow fibres is due to longer channel life-time. Preliminary results have been published elsewhere (Magazanik et al. 1979; Fedorov et al. 1981).
M e t h o d s
Preparations. Experiments were performed on the neuromus- cular junctions of m. cruralis and m. ileofibularis of the frog Rana temporaria (winter frogs, kept at 6 - 8 ~ C). The ventral (anterior) head of m. triceps femoris (m. cruralis) was dissected with the stump ofn. ischiadicus. This thin bundle is roughly triangular in shape and consists of fibres about 5 - 10ram length. Morphological examination and force measurements indicate that this muscle is relatively rich in typical slow fibres (Gilly 1975). Several experiments were done on fast fibres of re. sartorius and m. cutaneous pectoris.
Solutions. Bathing solution, of the following composition (raM): NaC1 115, KC1 2.5, CaC12 1.8, was buffered by
0031-6768/82/0394/0202/$01.80
203
NaHCO3 to a pH of 7.2. Temperature was measured by probe placed close to muscle and kept-at 2 0 - 2 2 ~ or at 8 -11 ~ (analysis of ACh-induced noise).
Drugs,. In some experiments cholinesterase inhibitors were used: neostigmine methylsulfate (prostigmine, Hoffmann-La Roche, Basel, Switzerland) permanently in the bathing so- lution at 3 x 10-6M o r the irreversible organophosphorous inhibitor armin (diethoxy-p-nitrophenyl phosphate), pre- liminary incubation for 30min in 5 x 10 7M. c~- Bungarotoxin (a generous gift by Drs. G. S. Tobias and M. A. Donlon, Washington University) was added to the bath in a final concentration 1 • 10 6 g/m1.
Voltage-Clamp System. The end-plate region was conven- tionally voltage clamped by two intracellular micropipettes. One of them was filled with 2.75 M KC1 and the other (current passing) electrode with 3 M potassium citrate. They had resistances of 3-10Mf2. The voltage clamping apparatus similar to that described by Dionne and Stevens (1975). Mepcs and ACh-induced noise were recorded as a voltage drop produced across a 1 MQ resistor in series with a current passing microelectrode. Membrane voltage was well con- trolled: the average voltage transient at the peak of the mepc was < 0.2 ~o of the net driving potential. The membrane potential was usually held at - 9 0 mV (fast fibres) and - 7 0 mV (slow fibres). The exact position of the clamping pipettes was checked by the sharpness of recorded miniature end-plate potentials ACh was applied ionophoretically close to the region between two micropipettes used for clamping. This system was also used to estimate the input resistance (Rin) and time constant (z) of rnuscle fibres. Pulses of current (0.2-2.0s duration) were passed through one microelec- trode, and electrotonic response recorded by the other.
Extracellular Recording. Glass micropipettes with broken and fire-polished end ( 5 - 1 0 gin) were filled with 1 M NaC1 + agar and placed close to the end-plate zones for extracellular recording of mepcs. Micropipettes with larger tip (inner diameter 15 -20 gm) were filled with Ringer solution con- taining 20 gM ACh. In this case the micropipettes served both for recording of synaptic currents and for application of ACh to postjunctional membrane (Neher and Sakmann 1976).
Analysis of ACh-Induced Noise. Fluctuations of synaptic current recorded in m. m. ileofibularis, sartorius and cu- taneous pectoris were digitized after appropriate filtering (0.5 -2,500 Hz) and analysed by F-37 signal analyzer (Soviet production). Sampling rate was 60 gs/point. Autocorrelation function (ACF), 512 points, was automatically calculated with maximal delay time 30.72ms. 512 to 1024 ACF were averaged. Averaged ACF of background noise was sub- tracted from averaged ACF obtained during ACh- application. The time constant of resultant ACF decay reflected the mean life-time (%oise) and the zero ordinate (in the case of voltage clamp) gave the mean channel con- ductance (7)-
Histochemical Techniques. The samples of muscle for his- tochemistry were frozen and serial transverse sections of m. cruralis stained for succinic dehydrogenase activity (SDH) according to the method of Stein and Padykula (1962). Lipid determinations were done according to Pearse (1972). The
same transverse sections were used for estimation of muscle fibre diameter with an ocular micrometer.
Results
A. Identification of Slow Muscle Fibres in M. cruralis
Two methods were used for identification of slow muscle fibres: electrophysiological and histochemical. It is known that the input resistance (Rin) and time constant (z) of slow muscle fibres are much higher than those of fast fibres (Burke and Ginsborg 1956; Adrian and Peachey 1965; Magazanik and Nasledov 1971). Fibres of the superficial layer of m. cruralis may be divided into 3 main classes: one which is characterized by rather low Rin (0.4-1.2 • 106ohm) and small z (3 - 20 ms), a second with high Rin (2.5 - 6 x 106 ohm) and large z (120-270ms) and a third with intermediate values of Rin and z. Among 103 superficial fibres studied 69 were "fast", 19 ~ were "slow" and 12 ~ were "intermediate". All slow fibres were unable to generate propagated action potentials. It was possible to reveal several discrete spots were mepcs are generated by moving the extracellular microelec- trode along the slow fibres.
Amphibian slow muscle fibres have rather low levels of succinate-dehydrogenase (SDH) activity and free lipid con- tent (Lannergren and Smith 1966). Investigation of the superficial layer of nine m. cruralis stained for SDH and lipids revealed on average 19~o (from 1 3 - 2 6 ~ in different mus- cles) light (slow) muscle fibres (Fig. 1). Muscle fibres poorly stained for SDH-activity are also characterized by larger diameter than typical fast fibres with high SDH-activity (Fig. 2). Thus there was a good correspondence between the electrophysiological and chemical features of different types of muscle fibres.
B. Spontaneous Miniature End-Plate Currents in Muscle Fibres of M. cruralis
Examples ofmepcs recorded at fast and slow muscle junctions are illustrated in Fig. 3. Mepcs in slow fibres are characterized by smaller mean amplitudes and slower time course. Typically, mepcs in all types of fibres decayed exponentially.
Estimates of mepc time course are summarized in Table 1. Mepc rise time (Tr) and half-time of decay (TI/2) were approximately 3 times longer in slow fibres than in fast ones. The results obtained with the voltage clamp method and by extracellular recording are in good agreement. No correlation between the rise time and half-time of decay was observed. Mepcs with sharp rise and slow decay as well as mepcs with rather long rise from zero to peak and moderate or slow rate of decay were observed. The decay half-time in slow fibres was also much more variable (Fig. 4A), with a distribution skewed towards higher values (Table2), than that in fast fibres which was narrow and rather symmetrical. However, there is little overlap between the two distributions.
C. Influence of Acetylcholinesterase (ACHE) Activity on the Time Course of Mepcs
Inhibition of AChE induced substantial prolongation of the mepc time course in all types of muscle fibres studied (Table 1, Fig. 4B). However, there are quantitative differences between the types of fibres. The more pronounced prolongation was revealed in fast fibres: here T r and T% increased 1.9 and 2.8
204
18
12
+!1 N i j
. . . . . . J % 1---]
B
z o io . . . . ,20 ~m
Fig. 2A and B. Histograms showing diameter of superficial fibres of m. cruralis stained for SDH. A Fast fibres; B slow fibres
Fig. 1 Transverse section of cruralis bundle surface stained for succinic dehydrogenase (SDH) activity. Dark fibres - fast, light ones - slow
~'ast
slow
Fig. 3. Miniature end-plate currents recorded extracellularly (left) and in voltage-clamped (right) fast and slow fibres of m. cruralis. Calibrations : vertical - I mV and 1 nA, horizontal - 2 ms
Table 1. Characteristics ofmepc's and of electrical constants of different types of muscle fibres. M. cruralis, Tf - rise-time ofmepc's, T,/2 - half-time of mepc decay, T membrane time constant, Rin - input resistance of fibre. Numbers give means _+ SE of all values obtained in individual fibres ; n number of fibres. More then 100 individual mepc's were estimated in each junction, t ~ = 2 0 - 2 3 ~
A. Voltage-clamp recording (holding potential - 9 0 mV in fast and - 70 mV in slow fibres)
Type of fibre n T r (ms) TI h (ms) Amplitude (hA) n z (ms) Rin x 106 ohm
Fig. 4A and B Histograms showing half-decay time of mepc's recorded under voltage- clamp in fast (hatched columns) and slow (blank columns) fibres of m. cruralis before (A) and after (B) acetylcholinesterase inhibition by neostigmine. A 1236 mepc's from 14 fast fibres, 676 mepc's from 7 slow fibres. B 638 mepc's from 7 fast fibres, 626 mepc's from 7 slow fibres
Table 2 Skewness coefficients a of distribution of mepc decay half-time in fast and slow muscle fibres under normal conditions, after ACHE- inhibition and after c~-bungarotoxin treatment. Mean • SE, number of fibres in brackets. P = level of significance
N.S. = not significant Z (xi-- k) 3
a Calculated as i f/~3
Type of AChE active fibre
AChE-inhibited
Control c~-BgTX P Control c~-BgTX P A treated B/A C treated D/C
times respectively; but in slow fibres, only 1.3 and 2.2 times. The difference between the kinetics of mepcs in fast and slow muscle fibres, remained significant however, with the time- constants differing by more than 2 times ( P < 0.001) even after AChE-inhibition.
The histograms of T,/~ showed prominent changes due to AChE-inhibition. The peaks shifted and distributions became more skewed. This phenomenon was more pronounced in slow fibres, but the coefficient of skewness (Table 2) shows that the fraction of slowly-decaying mepcs increased greatly in both types. Results obtained under voltage-clamp con- ditions and by extracellular recording are again in good agreement, giving practically identical ratios of TI/2 in both types of fibre.
D. Ejyects o f c~-Bungarotoxin on the Mepc Time Course
Prolongation of mepc time course by AChE-inhibi t ion may be explained by an increase of probability of repetitive binding of ACh-molecules to cholinoreceptors during the prolonged life-time of these molecules in the synaptic cleft.
One of the ways to decrease this probability and thereby to reduce the effect of anticholinesterase drugs on mepc time course is to decrease the density of the free receptors in the zone of ACh-quantum action (Katz and Miledi 1973).
The effect of c~-bungarotoxin on mepc time course in identified fast and slow fibres of m. ileofibularis was com- pared. Mepcs were recorded extracellularly in normal and armin-treated muscles. After sampling control mepcs c~- bungarotoxin was added to the bath in a final concentration 1 x 10 6 g/ml, and the changes in amplitude and time course mepcs were followed. As can be seen from Tables 2 and 3, and Fig. 5 this reduction of free cholinoreceptors induces not only the reduction of amplitudes but also the shortening of T,/,_ in both types of fibres. This is accompanied by distinct changes of distribution of individual mepc decay rate : the dispersion and the coefficient of assymetry are both reduced. All extremely slow mepcs disappear after c~-bungarotoxin treat- ment. These effects are more pronounced in slow than fast muscle fibres.
The shortening effect of ~-bungarotoxin on mepc decay in slow fibres was demonstrated not only in armin-treated
206
Table3. Effects of c~-bungarotoxin on the half-time of mepc decay recorded in fast and slow fibres ofm. ileofibularis before and after ACHE- inhibition (the effect of ~-bungarotoxin on mepc decay was measured after the mepc amplitude had diminished to 50 ~ of its initial level). Mean + SE in percent, in parenthesis - number of fibres; P = level of significance. Value of decay 71/~ before e-BgTX treatment taken as 100
Type of fibre AChE active AChE-inhibited
Fast 86 +_ 4 (3) 76 _+ 2 (9) N.S." P < 0.01 a
Slow 65 _+ 5 (4) 58 _+ 2 (6) P < 0.02 a P < 0.001 a
a Compared with control (before c~-BgTX treatment)
Y, 5C
0 s 10 i
1
~/ , , t ,_,__/ 5 10 15 20 ms
Fig. 5A and B. Histograms showing the effect of c~-bungarotoxin ( 1 x 10 - 6 g/ml) on the half-decay time of mepc's recorded extracellularly in fast (A) and slow (B) muscle fibres of m. ileofibularis treated with armin (J x J0 6M). Blank columns, control; hatched columns, during c~- bungarotoxin action. Note the disappearance of extremely slow mepc's in B during c~-bungarotoxin action. Each histogram includes not less than 600 mepc's estimated in 5 individual fibres
p repara t ion but also in normal ones : the difference be tween percentage values of shor tening obta ined in normal slow fibres and after AChE- inh ib i t i on was insignif icant (Table 3). In contras t ~-bungaro tox in had only a slight effect on the mepc t ime course in normal fast fibres (Tables 2 and 3).
E. Potential Dependency o f Mepc Decay Rate
Typical ly mepcs in all types of fibres decayed exponential ly . The decay r a t e I/'Cmepe was itself an exponent ia l funct ion o f vol tage (Fig. 6). In slow fibres in Tmepc was l inearly dependen t on vol tage only in the range - 60 to - 90 mV and appeared to saturate at more negat ive voltages, but l ineari ty persisted in fast fibres up to - 1 6 0 mV.
10-
-60 -90 -120 -150 mV
Fig. 6. Potential dependence of the time constant of mepc decay in voltage-clamped fast (triangles) and slow (squares) muscle fibres of m. ileofibularis. Mean _+ SD
Fast Fibre slow fibre
ACh
I i
Fig. 7. Examples of fluctuations of current recorded in voltage-clamped postjunctional membrane of fast and slow muscle fibres of the frog. Above - control noise; below - after application of acetylcholine. Calibrations : vertical - 2 nA, horizontal - 20 ms. Holding potential - 80mV; mean ACh-induced current 100nA; t ~ = 9~
F. Autocorrelation Function o f Acetylcholine-Induced Current Noise
The pat terns of current noise induced by appl ica t ion of A C h to the end-pla te region of fast and slow fibres of m. i leofibularis are demons t r a t ed in Fig. 7. The noise recorded in slow muscle fibres was more " r o u g h " . Au toco r r e l a t i on func t ion of noise ( A C F ) decayed exponent ia l ly and the rate o f decay did no t depend on the level of mean A C h - i n d u c e d current in all types of fibres studied. Howeve r this rate depended on the type of fibre (Fig. 8). It was possible to dist inguish at least three classes of junc t ions :
1. fast, where the l ife-t ime of synapt ic channels was ra ther shor t ; these junct ions be longed to s ingly- innervated fibres in the phasic par t of m. i leofibularis, m. sar tor ius and m. cu taneous pectoris.
2. slow, where the channel l ife-t ime was long - mult i - innerva ted fibres in the tonic pa r t o f m. ileofibularis.
3. in termedia te - s ingly- innervated fibres in the tonic pa r t of m. ileofibularis.
The equi l ib r ium potent ia l o f the A C h effect, as far as one can judge by ex t rapo la t ion of the cur ren t -vol tage curves, did no t differ significantly in the different types of fibres (esti- ma ted at - 5 mV). The synapt ic channels opened by A C h act ion in fast junc t ions had 1.5 t imes larger single channel conduc tance than in slow ones (Table 4).
10"
q {
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16 2
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�9 � 9 1 7 6 e �9
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L ~ 2 .6 a ~ �9 �9 �9 , \ ~ s.,
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0 3 6 9 T(rnsec) 12 15 18 21
207
F i g . 8
Autocorrelation functions of acetylcholine-induced noise recorded in different types of muscle fibres. Filled triangles - fast fibre; open triangles - intermediate fibre; filled circles - slow fibre. Abscissae: the delay time in ms; ordinate: autocorrelation function in A 2 (logarithmic scale). The lines were fitted using the least-square method. Figures near the lines are values of time constant of ACF decay. t ~ "= 8~ holding potential - 8 0 mV
Table4. Characteristics of the synaptic ACh-activated channels in different types of m. ileofibularis fibres obtained from the autocor- relation function of end-plate current fluctuations (%o~o), and time constant of decay of miniature end-plate currents recorded in the same fibres (Zmepo). t ' = + 8-- 11 ~ C; holding potential 80 mV; mean +_ SE; in parenthesis - number of fibres
Techniques Type of %o~e T m e p c Conductance fibre (ms) (ms) (pS)
Simultaneously with fluctuations induced by ACh, mepcs were sampled in the same junctions of m. ileofibularis. The mean time constant of mepc decay (rmepo) surpassed the mean life-time of ionic channels (Loise) in all types of fibres studied. In fast fibres this excess was rather small but in slow fibres "~mepc w a s 1.7-2.4 times larger than the channel life-time. Figure 9 demonstrated that in the fast fibres the mean life- time of channels coincides with the main peak of distribution of individual mepc decay time constants. In contrast, in slow fibres the mean life-time of ionic channels corresponded only with the decay time-constant of the fastest mepcs.
H. Potential and Temperature Dependence Of ZNois~
Channel life-time was dependent on the membrane potential in all types of muscle fibres. Z.oise increased with hyper- polarization and decreased with depolarization (Fig. 10). This dependence was exponential over the range - 4 0 to
- 120 mV, with a slightly smaller slope in slow fibres, r.oise was sensitive to temperature changes. Most experiments were
%
50 -
25
I
5 I r i
10 15 20 ms
Fig.9. Histograms of the time constant of mepc decay recorded in fast (hatched columns) and slow (blank columns) muscle fibres. Arrows show the values of the time constant of ACh-induced noise recorded in the same fibres, t ~ = 10 ~ C, holding potential - 8 0 mV
15
10
5
I I I I I -40 -80 -120 rnV
Fig. 10. Dependence of the time constant of ACh-induced noise on membrane potential in fast (squares) and slow (circles) muscle fibres; t ~ = 8~
208
performed at 8 - 1 1 ~ to reduce the mepc frequency, to slow down the onset of desensitization, and to shift the noise frequencies to the lower range, rnoiso at the same end-plate at 9 ~ and 20 ~ C was estimated in one experiment on a slow fibre, where it changed by 2.3 times.
I. Channels of Intermediate Type in Singly-Innervated Fibres
Attention should be drawn to the pronounced dispersion of %olsc values in individual slow multi-innervated fibres, which varied from 5.8 to 17.3 ms (mean value 8.3 ms). In fast fibres of m. ileofibularis the range of dispersion was more narrow (1.7 to 3.7 ms, mean value 2.9 ms) (Table 4). For comparison, estimations were performed under the same conditions in fast fibres from m. sartorius and m. cutaneous pectoris, giving mean values for Z,oisc of 2.9 _+ 0.2ms (n = 9) and 3.2 + 0.2 ms (n = 10), with approximately the same range of dispersion. Thus the values of life-time of ionic channels in typical frog fast fibres are in close agreement. However there were singly- innervated muscle fibres in tonic bundle of m. ileofibularis, where ~,oisr varied from 4.8 to 5.5ms (Fig. 8, Table4). As a rule, these fibres generated mepcs which decayed with a rate intermediate between the typical fast and slow fibres (section B of Results). These fibres can also generate propagated action potentials.
Discussion
Investigation of superficial fibres of m. cruralis revealed a rather large number of typical slow (tonic) muscle fibres. They are multiply innervated, generate only slow local responses, have higher input resistance and time constant, and are characterized by a low content of lipids and low activity of SDH. About 19 -20 % of the fibres studied are of this type. Other mixed frog muscles have a relatively small proportion of slow fibres in the superficial layer: the tonic bundle of m. ileofibularis has only single slow fibres (Adrian and Peachey I965; Nasledov and Fedorov 1965 ; Stefani and Steinbach 1969); m. piriformis, not more than 10~ (Stefani and Steinbach 1969). Thus m. cruralis is well suited for investigat- ing slow muscle fibres.
The main aim of the present study was to analyse the nature of the differences in mepc time course in fast and slow muscle fibres. Fidelity in recording these two kinds of mepcs may be limited by two different factors. The junction of fast fibres is extended but the space constant is relatively short. This leads to poor voltage-control of distinct parts of the junction (Anderson and Stevens 1973; Gage and McBurney 1975). On the other hand, the junction of slow fibre is compact and the space constant is relatively long, so that the postsynaptic membrane of these neuromuscular junctions should be effectively space-clamped. However, slow muscle fibres are innervated at several discrete sites. Combination of multiple innervation with long space constant may lead to recording of mepcs generated by several junctions although the recording electrodes were centered on only one (Dionne and Parsons 1981). Extracellular recording obviates the main sources of error discussed above. Agreement of results obtained by the two techniques suggests that neither limi- tation is too serious under voltage clamp.
The data show that mepcs in slow muscle fibres of m. cruralis differ from those in fast fibres in several ways:
1. the mean amplitude is rather less (by 1.3 times);
2. the rise time and half-time of decay are longer (in by 2 .2-2 .9 and 2 .8-3 .0 times correspondingly, depending on the mode of recording);
3. dispersion of the time course of individual mepcs is more pronounced.
An intermediate type of synaptic events was observed in some singly-innervated muscle fibres ofm. cruralis and tonic bundle of m. ileofibularis (Table 1 and 4).
These observations accord fully with earlier results on m. ileofibularis of the frog (Magazanik et al. 1979) and resemble in many respects the results obtained on pyriformis muscle of the frog (Miledi and Uchitel 1981) and slow muscle fibres of other species: snakes (Dionne and Parsons 1978, 1981), chick embryon (Rubin et al. 1979), adult chicken (Fedorov 1981), and rat (Magazanik et al. 1979; Fedorov 1980). They suggest a physiologically meaningful correlation between the functional properties of muscle fibres and the rates of synaptic response.
The time course of mepcs is determined by several processes :
1. interaction between free molecules of ACh released from nerve terminal and cholinoreceptors. (When AChE is active there is a low probability of repetitive binding of the same ACh molecule to a cholinoreceptor - Katz and Miledi 1975;)
2. conformational change of receptors from the inactive rest to the active conductive state;
3. rates of increasing and decreasing of ACh con- centration in the volume of synaptic cleft determined by diffusion of ACh-molecules;
4. enzymatic hydrolysis of ACh by acetylcholinesterase. The life-time of channels opened by ACh reflects the rates
of conformational change of cholinoreceptors. The distri- bution of estimated life-times of channels in different fibres, as obtained from the auto-correlation function (ACF) of ACh-induced noise, may be divided into 3 main classes (fast, slow and intermediate), which correspond well with the 3 types of fbres (fast singly-innervated, slow multi-innervated, and intermediate singly-innervated). The coexistence of chan- nels with different life-time in the region of former end-plate of denervated frog muscle fibre was shown earlier by obtain- ing ACF with a double exponential decay (Neher and Sakmann 1976). On the other hand ACFs of ACh-induced noise obtained in all muscle fibres studied here, including 38 typical slow ones, were well approximated by one exponent, suggesting a uniform population of channels in each type of fibre. However the resolution of the method of fluctuation analysis in the case of mixed channel population is rather low and depends particularly on the proportion of channels with different kinetics (Neher and Stevens 1977). We can conclude only that most channels in slow fibres are uniform and have slow kinetics.
It is now widely assumed that the rate of mepc decay normally reflect the rate at which receptors undergo a conformational change from open to dosed configuration (Magleby and Stevens 1972a, b). Some deviation from this rule may result from the retardation of the process of removal of ACh-molecules. As a consequence repetitive binding of ACh-molecules to receptors occurs and prolongs mepc decay (Katz and Miledi 1973; Magleby and Terrar 1975).
Our results suggest that the difference between mepc decay times is not due solely to differences in the life-time of channels opened by ACh in the various types of muscle fibres
209
(Table 4). In this respect our data differ from the observation by Miledi and Uchitel (1981) who have found a close agreement between channel life-time and time constant of decay of uni tary end-plate currents in slow muscle fibres. This difference in results may be due to some peculiarities of muscles (m. ileofibularis and m. pyriformis) or synaptic responses (mepc and uni tary end-plate currents induced by local applicat ion of Ca 2 + to only one junction). The present experiments have confirmed that the main (but not the only) factor governing the integral time course of mepcs in fast fibres is the channel open time. In slow muscle fibres, however, ~mepc was 1.7--2.4 times larger then ~no~se (both synaptic events were recorded in the same end-plate under the same experimental conditions), implying that in slow fibres addit ional factors to the longer channel life-time are needed to account for the mepc decay time constant. It seems that the relative importance of these factors differs greatly at different active spots, such that only the fastest mepcs in slow junctions are controlled by channels life-time (Fig. 3), and repetitive binding of ACh-molecules by receptors contributes greatly to the longer decay of the slower mepcs.
This was confirmed by analysis & t h e role of AChE in the time course of synaptic events in different types of muscle fibres. Inhibit ion of AChE by the irreversible organophos- phorous drug armin produced a more pronounced pro- longation of mepc decay in fast than in slow junctions. However the significant 2.2 times difference between the mean values of TI/2 in fast and slow fibres remained after AChE-inhibi t ion in m. cruralis, just as in earlier experiments on m. ileofibularis (Magazanik et al. 1979). On the other hand slowness of mepcs in embryonic (Rubin et al. 1979) or adult (Fedorov 1981) chicken slow muscles, and in rat slow muscles (Fedorov 1980) does seem to originate from a functional insufficiency of AChE in slow junctions. In these instances, the differences in the mepc decay rates between fast and slow fibres completely (chicken) or substantially (rat) disappeared after AChE-inhibi t ion.
Compar ison of the effects of , -bungaro toxin on the time course of mepcs provides further evidence in favour of a functional insufficiency of AChE in slow junctions. The significant shortening of mepc decay induced by , - bungarotoxin in fast fibres occurs only after AChE-inhibi t ion (Table 3, see also Katz and Miledi 1973; Magleby and Terrar 1975), whereas in slow fibres the degree of this effect did not depend on the activity of ACHE. This suggests that there are favourable condit ions in some slow junctions for repetitive binding of ACh-molecules to receptors even in the presence of intact ACHE. It seems that these conditions are nonuniform in different active spots of the slow junction. Thus a pronounced dispersion of Tmepc was revealed in both slow muscles m. ileofibularis and m. cruraSs. But after c~- bungarotoxin treatment the difference in dispersion of mepc decay in fast and slow junctions became insignificant (Table2). Non-uniformity may result from variat ion in AChE-act ivi ty and/or local peculiarities of morphology in different active spots of the slow junction (Magazanik et al. 1979).
The data obtained suggest also that there are obvious differences between properties of synaptic channels in muscle fibres of different functional types, Fur ther work is needed to elucidate the molecular nature of these differences. Nevertheless slow time course of synaptic response in slow frog fibres may be physiologically useful, because these fibres failed to generate a propagated action potential and the
contract ion is mainta ined by spatial and time summation of slow local synaptic potentials. Some propert ies of synaptic currents in slow muscle fibres (longer life-time of the open state, lower single channel conductance) resemble the extra- junct ional channels of denervated frog muscle (Neher and Sakmann 1976) or embryonic human and rat muscles (Bevan et al. 1978, 1979). Possibly the trophic influence of motor nerve controls not only the synthesis and distr ibution of cholinoreceptors but their inherent properties including the peculiarities of synaptic channel kinetics.
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