Brain Research, 88 (1975) 309-317 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
309
AN E L E C T R O P H Y S I O L O G I C A L E X A M I N A T I O N OF T H E C H A N G E S IN S K E L E T A L MUSCLE FIBRES IN RESPONSE TO D E G E N E R A T I N G NERVE TISSUE
ROSEMARY JONES* AND FRANTI~EK VYSKOt~IL Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Accepted November 25th, 1974)
SUMMARY Endplate free areas of the membrane of soleus and extensor digitorum longus muscles of the rat, covered for 3 days by a piece of nerve from the brachial region, become locally sensitive to ACh applied iontophoretically. Mean ACh sensitivity is increased at this site in the extensor muscle from O in areas not covered by the nerve to 6.27 mV/nanocoulombs (nC) ± 1.28 (S.E.), in the area under the nerve and in soleus from 0.12 mV/nC + 0.05 (S.E.) to 24.2 mV/nC + 4.6 (S.E.). Increase in sensitivity is accompanied by a decrease of resting membrane potential. Mean values are (unaffected areas) 75.0 mV -¢- 0.55 (S.E.) to (affected area) 65.0 mV + 0.56 (S.E.) in soleus and (unaffected areas) 76.3 mV + 0.61 (S.E.) to (affected area) 67.0 mV 4- 0.55 (S.E.) in extensor digitorum longus. These changes are local in individual fibres. Changes in ACh sensitivity and resting membrane potential in the area covered by the nerve are temporary, and after 6 days no sign of increase in sensitivity and drop in membrane potential were observed. The piece of nerve loses its potential to induce ACh sensitivity when it is heated to 98 °C for 5 min or soaked for 2 h in distilled water.
INTRODUCTION In vertebrate skeletal muscle sensitivity to acetylcholine (ACh) is restricted to the area of the fibre which is in closest contact with the motor nerve endings 1°,13,16. It is well known that the entire surface of the muscle fibres becomes sensitive to ACh after motor nerve section2,5,14. Sensitivity of the extrajunctional part of the muscle fibre membrane can, however, be induced by other stimuli such as mechanical damage 9, muscle inactivity7,12 or hibernation 19. * Present address: Department of Physiology, Medical School, Birmingham B15 2TJ, Great Britain.
310 It has been found 17,1s that a small piece of nerve, when placed onto the surfacc of the soleus muscle of a rat, induces an increase in the contracture response to applied ACh. Depolarisation of the fibres underlying the piece of nerve in response to ACh was also reported is. Using iontophoretic application of ACh and intracellular recording techniques we have examined in more detail the pattern of chemosensitivity in muscle fibres onto which a piece of nerve has been placed. METHODS
All experiments were performed at room temperature (20-22 °C) on soleus and extensor digitorum longus (EDL) muscles of Wistar rats of 200-250 g body weight.
Surgical procedure Under ether anaesthesia a piece of nerve about 2 mm in length was dissected from the brachial plexus of the rats and immersed in a dish containing sterile isotonic sodium chloride solution, while the wound under the forelimb was closed. An incision was then made along the lateral margin of the calf and the biceps femoris gently separated at the longitudinal tendon to expose the lateral margin of the soteus muscle. The piece of nerve was then carefully placed alongside the lower third of the soleus muscle (10 rats). This area is relatively free of synaptic connections. Further checks were made at the final experiment to ensure that the nerve lay only on endplate free areas of the muscle. In a further 6 rats a similar piece of nerve was placed onto the ventrolateral surface of the E D L muscle. In 4 rats the piece of brachial nerve was left in isotonic saline heated to 98 °C for 5 min before being put (after cooling) onto the soleus muscle. In a further 2 rats the piece of nerve was soaked in distilled water for 2 h at room temperature before the operation was completed. Unless otherwise stated, the EDL and soleus muscles were removed and their ACh sensitivity tested 3 days after the operation, i.e., at the time when the greatest increase of the contracture response was reported 8,1s.
Experimental procedure Operated muscles were dissected out and carefully cleaned of connective tissue in a bath containing a solution of the following composition (mM): Na +, 149.8; K +, 5.0; Ca 2+, 2.0; Mg 2+, 1.0; CI-, 147.8; HzPO4, 1.0; HCO~, 12.0; glucose, 11.01~, and oxygenated with a gas mixture of 95 % 02 and 5 % COz. The position of the piece of nerve was located under the dissecting microscope and a diagram of this area was drawn before the piece of nerve was removed to expose the underlying superficial muscle fibres for electrophysiological examination. In all cases it was observed that the piece of nerve had become rounded, and connective tissue and small blood vessels were present on the surface of the muscle in the area immediately surrounding it. Such features were incorporated in the diagram and provided landmarks by which the
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Fig. 1. Sensitivity of endplat¢ Dec parts of soleus muscle fibres 3 days operated to locally applied ACh. The inset shows a schematic representation of the muscle. A is the area underneath the piece of
brachial nerve - - the limit of this area is also indicated on the graph (broken vertical line) where the sensitivity of several fibres in this area are plotted (0, II, 0t, O). Two fibres were examined in another endplate free region of the muscle (B, O, N). These fibres show low 'background' sensitivity to ACh. Abscissa represents distance along the muscle fibres. Ordinate, ACh sensitivity expressed as mV/nC, log scale.
exact area occupied by the nerve could be recognised after its removal. The nerve was usually found to be loosely attached to the muscle and could easily be removed without damage to underlying fibres. Intracellular recording of changes in membrane potential were made using conventional glass micropipettes filled with 3 M KC1 and having a resistance of 10-15 Mf~ 4. A second pipette containing ACh (1 g/ml in water) was used for iontophoretic application of ACh, the technique being similar to that of Del Castillo and Katz a and Nastuk 16. The iontophoretic current was applied in pulses of 1-100 msec duration and monitored via a 50 Mf~ resistor in the circuit. Sensitivity to ACh was expressed as mV response per nanocoulomb (nC) charge applied to the ACh pipette 14. RESULTS
Sensitivity of operated soleus and EDL muscles to ACh In soleus fibres low background sensitivity comparable with that seen in unoperated rat soleus muscles 15 was observed. This sensitivity in no case exceeded 1 mV/nC and, as is indicated in the graph in Fig. 1 (open symbols corresponding to points in area B), is not evenly distributed along the membrane. When A C h and recording
312 pipettes were moved to the area of the muscle originally covered by the piece of nerve, high sensitivity to ACh was observed (filled symbols corresponding to points in area A in Fig. 1) exceeding background sensitivity by at least 200 times (see also Table I). This high sensitivity to ACh was observed in each of the 24 fibres examined. The area of high sensitivity usually covered 1.5-2 mm of muscle fibre length. Immediately outside this area responses to ACh were reduced, and at a distance of approximately 1 mm from the edge of the area previously occupied by the piece of nerve sensitivity was comparable with that found over the rest of the extrajunctional area of the muscle (see Fig. 1). Similar results were obtained when EDL muscles were examined. Sensitivity to ACh was observed over the area covered by the piece of nerve. However, maximum sensitivity recorded in EDL was never as great as in soleus (see Table I). Unlike soleus, the extrasynaptic regions of EDL fibres display no 'background' sensitivity to ACh. In all experiments ACh sensitivity was found over the entire area in contact with the piece of nerve, confirming the finding of Vrbov~d s. The sensitivity of single fibres was followed for distances of up to 2.8 mm of fibre length. Records obtained showed that the high sensitivity which was maintained over the area covered by the nerve declines in all areas immediately adjacent to this. As is apparent from Fig. 1 there is great variation in the absolute sensitivity recorded in different muscle fibres in the affected area. This might be explained by a difference in the ability of different fibres to respond to the influence of the nerve or simply by variations in the degree of contact of individual fibres with the nerve piece. The greater increase in sensitivity observed in soleus muscles compared with EDL (see Table I) may reflect a difference in the ability of these two muscles to respond to the stimulus. It is apparent from the results of experiments with denervated rat soleus and extensor muscles 1 that an increase in sensitivity on extrajunctional membrane of the same order can be obtained in both muscles. However, it is known that in soleus low background sensitivity can always be detected at extrajunctional membrane sites whereas the extrajunctional membrane of EDL is completely insensitive 15 (also present results). It may be that in an innervated muscle it is easier to induce the appearance of more ACh receptors when some receptors are already present. Areas of high chemosensitivity were not synaptic regions since no miniature endplate potentials (MEPPs) were recorded, and the area over which high sensitivity to ACh could be recorded was greater than that occupied by the endplate.
Sensitivity of fibres in deeper layers of the muscle It was of interest to see whether the observed increase in sensitivity in the area underlying the piece of nerve occurs only in superficial fibres, or whether fibres in deeper layers below the nerve also show hypersensitivity. The recording electrode was, therefore, inserted into fibres in a deeper (probably second) layer of the muscle, and ACh potentials recorded. Fig. 2 shows that in both soleus and EDL fibres lying beneath the superficial layer respond to ACh, although a pulse of about 500 msec is required to elicit this response. A long duration pulse is required because of the longer diffusion pathway from the ACh pipette to these deeper fibres. The lower traces in Fig. 2 indi-
313 SOL
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Fig. 2. Sensitivity of soleus (SOL) and EDL fibres from deeper (probably second) layer under the nerve (N). C: controls from fibres outside the affectedarea. cate that no such responses can be obtained in deeper layer fibres outside the affected region. Despite the long duration of the ACh pulse contractions of adjacent muscle fibres were not observed during ACh application to deeper fibres.
Duration of the sensitivity change The finding of Jones and Vrbov~ s, that the increase in sensitivity in muscles treated in this way reaches a peak at about 3 days after the operation and subsequently declines, was also confirmed. The mean sensitivity for 8 fibres in 2 soleus muscles examined 6 days after the operation was 0.29 mV/nC ± 0.13 (S.E.).
Effect of heating or osmotic shock on the ability of the piece of nerve to induce hypersensitivity To test whether only intact tissue is able to induce the increase in sensitivity, the piece of nerve was destroyed by placing it in saline heated to 98 °C or in distilled water for several minutes before placing it onto the soleus muscle. When these muscles were examined it was found that the area underlying the piece of nerve in these experiments showed only slightly increased sensitivity. The sensitivity of the fibres was measured at 3 spots over a distance of 0-500/~m of fibre length. Mean values for the maximum sensitivity recorded are 0.54 mV/nC d= 0.12 (S.E.) for 18 fibres in muscles with heated nerve and 0.36 ± 0.06 (S.E.) in 16 fibres covered by a piece of nerve soaked in distilled water.
Endplate sensitivity In most experiments the sensitivity of the synaptic area of several fibres was also measured. Mean values for both soleus (177 mV/nC) and EDL (203 mV/nC) were always several times greater than the maximum sensitivity observed under the piece of degenerating nerve (see Table I).
Resting membrane potentials Throughout the experiments the value of the recorded resting membrane po-
314 TABLE I SOLEUSAND EDL 3 DAYSAFTEROPERATION Mean values for sensitivity and resting membrane potential (RMP) of fibres measured in the area under nerve (area A) and control area (area B). Figures in brackets ~ number of fibres.
A Ch sensitivity
RMP
(mV/nC) Soleus
Area under nerve (24) Control area (15) At endplate (7)
24.2 ~ 4.6 0.12 4- 0.05 177.0 4- 31.10
65.1 4- 0.56 75 ~: 0.55 73.4 4- 0.75
6.27 4- 1.28 203.3 ± 28.1
67.0 ± 0.55 75.8 4- 0.30
EDL
Area under nerve (23) At endplate (6)
tential (RMP) was noted each time the pipette was inserted into a fibre. It was seen that in the area of high sensitivity, the R M P was lower than that recorded elsewhere. Mean values for the R M P of fibres covered by the nerve and those outside the affected area are given in Table I. These figures show a considerable difference in R M P in both soleus and E D L fibres in areas showing high sensitivity to ACh, compared with unaffected areas of the same muscle. That lower resting membrane potentials were found on the areas of fibres that showed high A C h sensitivity is further illustrated by the results shown in Fig. 3. The figure shows plots of sensitivity and traces of evoked responses from 6 soleus muscle fibres, as the electrodes were moved laterally across the area occupied by the nerve. The inset above indicates the sites at which recordings were made, and the observed R M P for each fibre is indicated next to each trace. Thus, both the change in sensitivity and lower resting membrane potential are restricted to areas of the fibre underlying the piece of nerve. Six days after the operation, when sensitivity has returned to near normal values, R M P was also found to be restored. Mean value in 8 fibres in the area under the nerve in 2 soleus muscles was 73 mV ± 1.73 (S.E.). In experiments in which the nerve was destroyed, and no increase in sensitivity was seen, resting membrane potentials were near normal, mean values being 73.37 ± 0.31 (S.E.) in 18 fibres with heated nerve and 73.0 ~: 0.45 (S.E.) in 16 fibres with nerve that had been subjected to osmotic shock. DISCUSSION
It is clear from these results that the increase in sensitivity observed when a piece of nerve is placed onto the surface of an innervated muscle is local to the area in contact with the nerve, and that the properties of the rest of the muscle fibre membrane are normal. The induction of a discrete area of high chemosensitivity on the area of the
315
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Fig. 3. The sensitivity of individual soleus muscle fibres to ACh across the area covered by the piece of nerve, eD and ~ : fibres just at the edge of this area. Upper insert: schematic diagram of the area under investigation. Limit of the area covered by the nerve indicated by continuous line. Right insert: examples of ACh potentials obtained at points on the graph. Resting membrane potentials are shown. Only 2-3 places on each fibre were examined. muscle fibre membrane underlying a piece of degenerating nerve is similar to the situation existing at the endplate, where only the area of the membrane in contact with the motor nerve is sensitive to the transmitter. The way in which the synaptic area of the muscle is kept sensitive to the transmitter while all other areas of the normal muscle fibre show little or no chemosensitivity is not clear. In view of the present results, however, it may be suggested that contact with nerve tissue brings about changes in the muscle fibre membrane causing it to become sensitive to ACh. This may be as a result of the action of substances released by the nerve axon, or in response to the presence of degenerating nerve tissue. At the neuromuscular junction substances released from the axon terminal, or as a result of nerve terminal degeneration 8 may ensure the high chemosensitivity of the postjunctional membrane. The increase in sensitivity in response to the piece of nerve is transient. Six days after the operation only low sensitivity is seen in the area underlying the nerve. This is due, perhaps, to an exhaustion of the sensitising influence in the nerve piece.Receptors already present apparently disappear, and this may be because these muscles are innervated and consequently retain their normal pattern of activity. Muscle activity is known to reduce hypersensitivity in denervated muscles s,12, and previous experiments la have shown that action potential conduction through the region of high sensitivity remains unaltered during the period of changing sensitivity. The mechanism by which high sensitivity is induced by the piece of nerve is not indicated by the present experiments, but different possibilities may be considered. It may be that receptors are produced de novo on this part of the muscle fibre membrane.
316 Factors originating in the nerve may locally induce the synthesis of ACh receptors. If this is the case, then the development of receptors should be inhibited by drugs which block the translational stage of protein synthesis, as has been found in denervated muscles 6. On the other hand, it has been suggested that increase in muscle sensitivity following denervation or muscle damage may be due to 'unmasking' receptors already present in the membrane s. Such a process may be induced by the presence of the piece of nerve. The increase in sensitivity can be attributed to factors acting immediately in the vicinity of the nerve, since all other areas of the muscle were found to be unaffected by the procedure. It is thought, therefore, that the observed changes are not due to muscle damage, since the hypersensitivity resulting from trauma is less well localised and other changes in the properties of the muscle fibres are observed 9. In the present experiments nerves which had been heated or subjected to osmotic shock failed to induce such a marked response, indicating that the procedure alone is not responsible for inducing the observed changes. Histological examination of transverse sections of the region of the muscle occupied by the piece of nerve revealed no differences in the degree of inflammatory changes caused by intact or heated pieces of nerve (to be published). However, this does not indicate whether the activity of cells involved in such an inflammatory reaction is different depending on whether the nerve is heated or not. Further experiments are required to elucidate this point. ACKNOWLEDGEMENT
R. Jones would like to thank the Royal Society for financial support which enabled her to visit the Institute of Physiology, Czechoslovak Academy of Sciences, in Prague, where this work was carried out.
REFERENCES 1 ALBUQUERQUE,E. X., AND MCISAAC, R. J., Early development of acetylcholine receptors on fast
and slow mammalian skeletal muscle, Life Sci., 8 (1969) 409-416. 2 AXELSSON,J., AND Tt-~SLEFF,S., A study of supersensitivity in denervated mammalian skeletal muscle, J. Physiol. (Lond.), 147 (1959) 178-193. 3 DEE CASTILLO,J., ANt)KATZ,B., Quantal components of the endplate potential, 3".Physiol. (Lond.), 124 (1954) 560-573. 4 FATT, P., AND KATZ, B., An analysis of the endplate potential recorded with an intra-cellular electrode, J. Physiol. (Lond.), 115 (1951) 320-370. 5 GINETZINSKI,A. G., AND SHAMARINA,N. M., The tonomotor phenomenon in denervated muscle,
Usp. sovrem. Biol., 15 (1942) 283-294. 6 GRAMPP,W., HARRIS,J. B., AND THESLEFF,S., Inhibition of denervation changes in skeletal muscle
by blockers of protein synthesis, J. Physiol. (Lond.), 221 (1972) 743-754. 7 JOHNS,T. R., ANDTH~SLErF,S., Effects of motor inactivation on the chemical sensitivity of skeletal muscle, Acta physiol, scand., 51 (1961) 136-141. 8 JONES,R., AND VRBOV•, G,, Two factors responsible for the development of denervation hypersensitivity, J. Physiol. (Lond.), 236 (1974) 517-538. 9 KATZ,B., ANDMILEDI,R., The development of ACh sensitivity in nerve-free segments of skeletal muscle, J. Physiol. (Lond.), 170 (1964) 389-396.
317 10 KUFFLER,S. W., Specific excitability of the endplate region in normal and denervated muscle, 3.. NeurophysioL, 6 (1943) 99-110. 11 LILEY,A. W., An investigation of spontaneous activity of the neuromuscular junction of the rat, J. Physiol. (Lond.), 132 (1956) 650--666. 12 LOMO,T., AND ROSENTHAL,J., Control of ACh sensitivity by muscle activity in the rat, J. Physiol. (Lond.), 221 (1972) 493-513. 13 MIL~DI, R., Junctional and extrajunctional acetylcholine receptors in skeletal muscle fibers, J. Physiol. (Lond.), 151 (1960) 24-30. 14 MILEDI, R., The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1-23. 15 MILEDI, R., AND ZELEN~,,J., Sensitivity to acetylcholine in rat slow muscle, Nature (Lond.), 210 (1966) 855-856. 16 NASTUK,W. L., Membrane potential changes at single muscle endplate produced by transitory application of acetylcholine with an electrically controlled microjet, Fed. Proc., 12 (1953) 102. 17 VRBOV~,,G., Induction of an extrajunctional chemosensitive area in intact innervated muscle fibres, J. Physiol. (Lond.), 191 (1967) 20-21 P. 18 VRBOVA,G., Control of chemosensitivity at the neuromuscular junction. In R. EI~ENMANtq(Ed.), Proc. 4th int. Congr. Pharmacol., Vol. 111, Schwabe and Co., Basel, 1970, pp. 158-169. 19 VYSKOt~m,F., MORAVEC,J., ANDJANSK'?,L., Resting state of the myoneural junction in a hibernator, Brain Research, 34 (1971) 381-384.
309
AN E L E C T R O P H Y S I O L O G I C A L E X A M I N A T I O N OF T H E C H A N G E S IN S K E L E T A L MUSCLE FIBRES IN RESPONSE TO D E G E N E R A T I N G NERVE TISSUE
ROSEMARY JONES* AND FRANTI~EK VYSKOt~IL Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Accepted November 25th, 1974)
SUMMARY Endplate free areas of the membrane of soleus and extensor digitorum longus muscles of the rat, covered for 3 days by a piece of nerve from the brachial region, become locally sensitive to ACh applied iontophoretically. Mean ACh sensitivity is increased at this site in the extensor muscle from O in areas not covered by the nerve to 6.27 mV/nanocoulombs (nC) ± 1.28 (S.E.), in the area under the nerve and in soleus from 0.12 mV/nC + 0.05 (S.E.) to 24.2 mV/nC + 4.6 (S.E.). Increase in sensitivity is accompanied by a decrease of resting membrane potential. Mean values are (unaffected areas) 75.0 mV -¢- 0.55 (S.E.) to (affected area) 65.0 mV + 0.56 (S.E.) in soleus and (unaffected areas) 76.3 mV + 0.61 (S.E.) to (affected area) 67.0 mV 4- 0.55 (S.E.) in extensor digitorum longus. These changes are local in individual fibres. Changes in ACh sensitivity and resting membrane potential in the area covered by the nerve are temporary, and after 6 days no sign of increase in sensitivity and drop in membrane potential were observed. The piece of nerve loses its potential to induce ACh sensitivity when it is heated to 98 °C for 5 min or soaked for 2 h in distilled water.
INTRODUCTION In vertebrate skeletal muscle sensitivity to acetylcholine (ACh) is restricted to the area of the fibre which is in closest contact with the motor nerve endings 1°,13,16. It is well known that the entire surface of the muscle fibres becomes sensitive to ACh after motor nerve section2,5,14. Sensitivity of the extrajunctional part of the muscle fibre membrane can, however, be induced by other stimuli such as mechanical damage 9, muscle inactivity7,12 or hibernation 19. * Present address: Department of Physiology, Medical School, Birmingham B15 2TJ, Great Britain.
310 It has been found 17,1s that a small piece of nerve, when placed onto the surfacc of the soleus muscle of a rat, induces an increase in the contracture response to applied ACh. Depolarisation of the fibres underlying the piece of nerve in response to ACh was also reported is. Using iontophoretic application of ACh and intracellular recording techniques we have examined in more detail the pattern of chemosensitivity in muscle fibres onto which a piece of nerve has been placed. METHODS
All experiments were performed at room temperature (20-22 °C) on soleus and extensor digitorum longus (EDL) muscles of Wistar rats of 200-250 g body weight.
Surgical procedure Under ether anaesthesia a piece of nerve about 2 mm in length was dissected from the brachial plexus of the rats and immersed in a dish containing sterile isotonic sodium chloride solution, while the wound under the forelimb was closed. An incision was then made along the lateral margin of the calf and the biceps femoris gently separated at the longitudinal tendon to expose the lateral margin of the soteus muscle. The piece of nerve was then carefully placed alongside the lower third of the soleus muscle (10 rats). This area is relatively free of synaptic connections. Further checks were made at the final experiment to ensure that the nerve lay only on endplate free areas of the muscle. In a further 6 rats a similar piece of nerve was placed onto the ventrolateral surface of the E D L muscle. In 4 rats the piece of brachial nerve was left in isotonic saline heated to 98 °C for 5 min before being put (after cooling) onto the soleus muscle. In a further 2 rats the piece of nerve was soaked in distilled water for 2 h at room temperature before the operation was completed. Unless otherwise stated, the EDL and soleus muscles were removed and their ACh sensitivity tested 3 days after the operation, i.e., at the time when the greatest increase of the contracture response was reported 8,1s.
Experimental procedure Operated muscles were dissected out and carefully cleaned of connective tissue in a bath containing a solution of the following composition (mM): Na +, 149.8; K +, 5.0; Ca 2+, 2.0; Mg 2+, 1.0; CI-, 147.8; HzPO4, 1.0; HCO~, 12.0; glucose, 11.01~, and oxygenated with a gas mixture of 95 % 02 and 5 % COz. The position of the piece of nerve was located under the dissecting microscope and a diagram of this area was drawn before the piece of nerve was removed to expose the underlying superficial muscle fibres for electrophysiological examination. In all cases it was observed that the piece of nerve had become rounded, and connective tissue and small blood vessels were present on the surface of the muscle in the area immediately surrounding it. Such features were incorporated in the diagram and provided landmarks by which the
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Fig. 1. Sensitivity of endplat¢ Dec parts of soleus muscle fibres 3 days operated to locally applied ACh. The inset shows a schematic representation of the muscle. A is the area underneath the piece of
brachial nerve - - the limit of this area is also indicated on the graph (broken vertical line) where the sensitivity of several fibres in this area are plotted (0, II, 0t, O). Two fibres were examined in another endplate free region of the muscle (B, O, N). These fibres show low 'background' sensitivity to ACh. Abscissa represents distance along the muscle fibres. Ordinate, ACh sensitivity expressed as mV/nC, log scale.
exact area occupied by the nerve could be recognised after its removal. The nerve was usually found to be loosely attached to the muscle and could easily be removed without damage to underlying fibres. Intracellular recording of changes in membrane potential were made using conventional glass micropipettes filled with 3 M KC1 and having a resistance of 10-15 Mf~ 4. A second pipette containing ACh (1 g/ml in water) was used for iontophoretic application of ACh, the technique being similar to that of Del Castillo and Katz a and Nastuk 16. The iontophoretic current was applied in pulses of 1-100 msec duration and monitored via a 50 Mf~ resistor in the circuit. Sensitivity to ACh was expressed as mV response per nanocoulomb (nC) charge applied to the ACh pipette 14. RESULTS
Sensitivity of operated soleus and EDL muscles to ACh In soleus fibres low background sensitivity comparable with that seen in unoperated rat soleus muscles 15 was observed. This sensitivity in no case exceeded 1 mV/nC and, as is indicated in the graph in Fig. 1 (open symbols corresponding to points in area B), is not evenly distributed along the membrane. When A C h and recording
312 pipettes were moved to the area of the muscle originally covered by the piece of nerve, high sensitivity to ACh was observed (filled symbols corresponding to points in area A in Fig. 1) exceeding background sensitivity by at least 200 times (see also Table I). This high sensitivity to ACh was observed in each of the 24 fibres examined. The area of high sensitivity usually covered 1.5-2 mm of muscle fibre length. Immediately outside this area responses to ACh were reduced, and at a distance of approximately 1 mm from the edge of the area previously occupied by the piece of nerve sensitivity was comparable with that found over the rest of the extrajunctional area of the muscle (see Fig. 1). Similar results were obtained when EDL muscles were examined. Sensitivity to ACh was observed over the area covered by the piece of nerve. However, maximum sensitivity recorded in EDL was never as great as in soleus (see Table I). Unlike soleus, the extrasynaptic regions of EDL fibres display no 'background' sensitivity to ACh. In all experiments ACh sensitivity was found over the entire area in contact with the piece of nerve, confirming the finding of Vrbov~d s. The sensitivity of single fibres was followed for distances of up to 2.8 mm of fibre length. Records obtained showed that the high sensitivity which was maintained over the area covered by the nerve declines in all areas immediately adjacent to this. As is apparent from Fig. 1 there is great variation in the absolute sensitivity recorded in different muscle fibres in the affected area. This might be explained by a difference in the ability of different fibres to respond to the influence of the nerve or simply by variations in the degree of contact of individual fibres with the nerve piece. The greater increase in sensitivity observed in soleus muscles compared with EDL (see Table I) may reflect a difference in the ability of these two muscles to respond to the stimulus. It is apparent from the results of experiments with denervated rat soleus and extensor muscles 1 that an increase in sensitivity on extrajunctional membrane of the same order can be obtained in both muscles. However, it is known that in soleus low background sensitivity can always be detected at extrajunctional membrane sites whereas the extrajunctional membrane of EDL is completely insensitive 15 (also present results). It may be that in an innervated muscle it is easier to induce the appearance of more ACh receptors when some receptors are already present. Areas of high chemosensitivity were not synaptic regions since no miniature endplate potentials (MEPPs) were recorded, and the area over which high sensitivity to ACh could be recorded was greater than that occupied by the endplate.
Sensitivity of fibres in deeper layers of the muscle It was of interest to see whether the observed increase in sensitivity in the area underlying the piece of nerve occurs only in superficial fibres, or whether fibres in deeper layers below the nerve also show hypersensitivity. The recording electrode was, therefore, inserted into fibres in a deeper (probably second) layer of the muscle, and ACh potentials recorded. Fig. 2 shows that in both soleus and EDL fibres lying beneath the superficial layer respond to ACh, although a pulse of about 500 msec is required to elicit this response. A long duration pulse is required because of the longer diffusion pathway from the ACh pipette to these deeper fibres. The lower traces in Fig. 2 indi-
313 SOL
EDL
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I
IO'7A
Fig. 2. Sensitivity of soleus (SOL) and EDL fibres from deeper (probably second) layer under the nerve (N). C: controls from fibres outside the affectedarea. cate that no such responses can be obtained in deeper layer fibres outside the affected region. Despite the long duration of the ACh pulse contractions of adjacent muscle fibres were not observed during ACh application to deeper fibres.
Duration of the sensitivity change The finding of Jones and Vrbov~ s, that the increase in sensitivity in muscles treated in this way reaches a peak at about 3 days after the operation and subsequently declines, was also confirmed. The mean sensitivity for 8 fibres in 2 soleus muscles examined 6 days after the operation was 0.29 mV/nC ± 0.13 (S.E.).
Effect of heating or osmotic shock on the ability of the piece of nerve to induce hypersensitivity To test whether only intact tissue is able to induce the increase in sensitivity, the piece of nerve was destroyed by placing it in saline heated to 98 °C or in distilled water for several minutes before placing it onto the soleus muscle. When these muscles were examined it was found that the area underlying the piece of nerve in these experiments showed only slightly increased sensitivity. The sensitivity of the fibres was measured at 3 spots over a distance of 0-500/~m of fibre length. Mean values for the maximum sensitivity recorded are 0.54 mV/nC d= 0.12 (S.E.) for 18 fibres in muscles with heated nerve and 0.36 ± 0.06 (S.E.) in 16 fibres covered by a piece of nerve soaked in distilled water.
Endplate sensitivity In most experiments the sensitivity of the synaptic area of several fibres was also measured. Mean values for both soleus (177 mV/nC) and EDL (203 mV/nC) were always several times greater than the maximum sensitivity observed under the piece of degenerating nerve (see Table I).
Resting membrane potentials Throughout the experiments the value of the recorded resting membrane po-
314 TABLE I SOLEUSAND EDL 3 DAYSAFTEROPERATION Mean values for sensitivity and resting membrane potential (RMP) of fibres measured in the area under nerve (area A) and control area (area B). Figures in brackets ~ number of fibres.
A Ch sensitivity
RMP
(mV/nC) Soleus
Area under nerve (24) Control area (15) At endplate (7)
24.2 ~ 4.6 0.12 4- 0.05 177.0 4- 31.10
65.1 4- 0.56 75 ~: 0.55 73.4 4- 0.75
6.27 4- 1.28 203.3 ± 28.1
67.0 ± 0.55 75.8 4- 0.30
EDL
Area under nerve (23) At endplate (6)
tential (RMP) was noted each time the pipette was inserted into a fibre. It was seen that in the area of high sensitivity, the R M P was lower than that recorded elsewhere. Mean values for the R M P of fibres covered by the nerve and those outside the affected area are given in Table I. These figures show a considerable difference in R M P in both soleus and E D L fibres in areas showing high sensitivity to ACh, compared with unaffected areas of the same muscle. That lower resting membrane potentials were found on the areas of fibres that showed high A C h sensitivity is further illustrated by the results shown in Fig. 3. The figure shows plots of sensitivity and traces of evoked responses from 6 soleus muscle fibres, as the electrodes were moved laterally across the area occupied by the nerve. The inset above indicates the sites at which recordings were made, and the observed R M P for each fibre is indicated next to each trace. Thus, both the change in sensitivity and lower resting membrane potential are restricted to areas of the fibre underlying the piece of nerve. Six days after the operation, when sensitivity has returned to near normal values, R M P was also found to be restored. Mean value in 8 fibres in the area under the nerve in 2 soleus muscles was 73 mV ± 1.73 (S.E.). In experiments in which the nerve was destroyed, and no increase in sensitivity was seen, resting membrane potentials were near normal, mean values being 73.37 ± 0.31 (S.E.) in 18 fibres with heated nerve and 73.0 ~: 0.45 (S.E.) in 16 fibres with nerve that had been subjected to osmotic shock. DISCUSSION
It is clear from these results that the increase in sensitivity observed when a piece of nerve is placed onto the surface of an innervated muscle is local to the area in contact with the nerve, and that the properties of the rest of the muscle fibre membrane are normal. The induction of a discrete area of high chemosensitivity on the area of the
315
5 0 m sec
RMP
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66
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67
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74
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Fig. 3. The sensitivity of individual soleus muscle fibres to ACh across the area covered by the piece of nerve, eD and ~ : fibres just at the edge of this area. Upper insert: schematic diagram of the area under investigation. Limit of the area covered by the nerve indicated by continuous line. Right insert: examples of ACh potentials obtained at points on the graph. Resting membrane potentials are shown. Only 2-3 places on each fibre were examined. muscle fibre membrane underlying a piece of degenerating nerve is similar to the situation existing at the endplate, where only the area of the membrane in contact with the motor nerve is sensitive to the transmitter. The way in which the synaptic area of the muscle is kept sensitive to the transmitter while all other areas of the normal muscle fibre show little or no chemosensitivity is not clear. In view of the present results, however, it may be suggested that contact with nerve tissue brings about changes in the muscle fibre membrane causing it to become sensitive to ACh. This may be as a result of the action of substances released by the nerve axon, or in response to the presence of degenerating nerve tissue. At the neuromuscular junction substances released from the axon terminal, or as a result of nerve terminal degeneration 8 may ensure the high chemosensitivity of the postjunctional membrane. The increase in sensitivity in response to the piece of nerve is transient. Six days after the operation only low sensitivity is seen in the area underlying the nerve. This is due, perhaps, to an exhaustion of the sensitising influence in the nerve piece.Receptors already present apparently disappear, and this may be because these muscles are innervated and consequently retain their normal pattern of activity. Muscle activity is known to reduce hypersensitivity in denervated muscles s,12, and previous experiments la have shown that action potential conduction through the region of high sensitivity remains unaltered during the period of changing sensitivity. The mechanism by which high sensitivity is induced by the piece of nerve is not indicated by the present experiments, but different possibilities may be considered. It may be that receptors are produced de novo on this part of the muscle fibre membrane.
316 Factors originating in the nerve may locally induce the synthesis of ACh receptors. If this is the case, then the development of receptors should be inhibited by drugs which block the translational stage of protein synthesis, as has been found in denervated muscles 6. On the other hand, it has been suggested that increase in muscle sensitivity following denervation or muscle damage may be due to 'unmasking' receptors already present in the membrane s. Such a process may be induced by the presence of the piece of nerve. The increase in sensitivity can be attributed to factors acting immediately in the vicinity of the nerve, since all other areas of the muscle were found to be unaffected by the procedure. It is thought, therefore, that the observed changes are not due to muscle damage, since the hypersensitivity resulting from trauma is less well localised and other changes in the properties of the muscle fibres are observed 9. In the present experiments nerves which had been heated or subjected to osmotic shock failed to induce such a marked response, indicating that the procedure alone is not responsible for inducing the observed changes. Histological examination of transverse sections of the region of the muscle occupied by the piece of nerve revealed no differences in the degree of inflammatory changes caused by intact or heated pieces of nerve (to be published). However, this does not indicate whether the activity of cells involved in such an inflammatory reaction is different depending on whether the nerve is heated or not. Further experiments are required to elucidate this point. ACKNOWLEDGEMENT
R. Jones would like to thank the Royal Society for financial support which enabled her to visit the Institute of Physiology, Czechoslovak Academy of Sciences, in Prague, where this work was carried out.
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317 10 KUFFLER,S. W., Specific excitability of the endplate region in normal and denervated muscle, 3.. NeurophysioL, 6 (1943) 99-110. 11 LILEY,A. W., An investigation of spontaneous activity of the neuromuscular junction of the rat, J. Physiol. (Lond.), 132 (1956) 650--666. 12 LOMO,T., AND ROSENTHAL,J., Control of ACh sensitivity by muscle activity in the rat, J. Physiol. (Lond.), 221 (1972) 493-513. 13 MIL~DI, R., Junctional and extrajunctional acetylcholine receptors in skeletal muscle fibers, J. Physiol. (Lond.), 151 (1960) 24-30. 14 MILEDI, R., The acetylcholine sensitivity of frog muscle fibres after complete or partial denervation, J. Physiol. (Lond.), 151 (1960) 1-23. 15 MILEDI, R., AND ZELEN~,,J., Sensitivity to acetylcholine in rat slow muscle, Nature (Lond.), 210 (1966) 855-856. 16 NASTUK,W. L., Membrane potential changes at single muscle endplate produced by transitory application of acetylcholine with an electrically controlled microjet, Fed. Proc., 12 (1953) 102. 17 VRBOV~,,G., Induction of an extrajunctional chemosensitive area in intact innervated muscle fibres, J. Physiol. (Lond.), 191 (1967) 20-21 P. 18 VRBOVA,G., Control of chemosensitivity at the neuromuscular junction. In R. EI~ENMANtq(Ed.), Proc. 4th int. Congr. Pharmacol., Vol. 111, Schwabe and Co., Basel, 1970, pp. 158-169. 19 VYSKOt~m,F., MORAVEC,J., ANDJANSK'?,L., Resting state of the myoneural junction in a hibernator, Brain Research, 34 (1971) 381-384.
