Synchronization of potentials and response to direct current stimulation in denervated mammalian muscle

SYNCHRONIZATION OF POTENTIALS AND RESPONSE TO DIRECT CURRENT STIMULATION IN DENERVATED MAMMALIAN MUSCLE WILLIAM

M. LANDAU,

M.D. 1

Ldboratory of NeurophyJiology, Department of Neuropsychiatry, University School of Medicine. St, Louis. MO.

INTRODUCTION in relation to an electromyographic study of clinical myotonia (Lanldau 1951) some related observations upon denervated muscle have been made, which are reported here. A similar response to direct current in denervated and ,myotonic muscle was noted first by Erb (1886) and more recently by Bourguignon (1923). Ravin (1940) demonstrated a “warming up” phenomenon in denerva,ted muscle stimulated with short shocks analogous to the decrease of clinical myotonia with exercise, and has called abteation to pharmacological and histological similarities. Denny-Brown and Nevin (1941) observed that the fine potential electromyographic record of “peripheral” myotoaia is indistinguishable from that of denervated muscle (Denny-Brown and Pennybacker 1938). Recently Pollock et al. (1946), described the behavior of denetvated muscle in response to various types of stimulating current. Doupe (1943) and Kugelberg and Petersen (1949) demonstrated iterative spiLe potentials led from denervated human muscle direct current sti,mulation. Synchronization in denervated muscle was observed by Harvey and Kuffler (1944)) Weddell et al. (1944), Kugelberg and Petersen (1949) and Jasper and Ballem (1949) _ The initial purpose of this study was to make a detailed investigation of these phenomena. Observations have been incltuded of resting * activity and of activity after administ,ration of drugs in denervated muscle. Mechanically induced aotivity in normal and denervated musde was studied. METHODS Adult rabbits ponse to electrical

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milarly to various mammalian species including man (Pollock et a/. 1946), (Feinstein et al. 1945). Operations and electromyographic observations were made under Nembutal anesthesia. Denervation was accomplished by section of the sciatic nerve above the greater Itrochanter with turning back of the cut ends. Inspection at the time of death showed no evidence of reinnervation. The gastrocnemius and other leg muscles were studied using needle electrodes placed percutaneously. Obsantions were made from the acute stage to the 108th day after nerve section. The animals were fastened in prone ,Position to an animal board with ankle ties and a band around the lower back. Artificial respiration was administered through a tracheal canula when the animals were curarized. Curare and Prostigmine were administered intravenously. The degree of neuromuscular block was tested by sti,muIation of the sciatic isolated on the side opposite that being examined. Polarization was carried out using 1 cm. saline pad electrodes fixed to the skin with adhesive tape, one of which was in the popliteal fossa and the other over the lower portion of the gastrocnemius tendon. A dry cell battery in series with a 2000 ohm resistance supplied the current; “noise” was negligible at the lower voltages. Current was not measured dire&y. The electrode-tissue resistance measured with a Wheatstone Bridge was approximately 800 to 1000 ohms. A Wagner ground in the stimulating circuit aided in adjusting for shock artifact. Thresholds for palpable and visual responses were recorded in many experiments. Bipolar needle electrodes with a grounded shaft were used. They consisted of two no. 40 (75 I(.) Nichrome wires threaded thrqugh a no. 24 or no. 25 hypodermic needle, embedded in methacrylate plastic, and then ground smooth. The tips were 10 to 400 p apart. Such electrodes give most critical localization of leading off (Adrian and Bronk 1929; tindsley 1935; Brown 1937a; Petersen and Kugelberg 1949), and proved particularly advantageous for successful recording from near the site of electrical stimulation. Satisfactory fixation for prolonged recording from a single locus was obtained by attaching the stiff needle leads to the animal board with adhesive tape. Potentials were amplified with a Grass amplifier and oscillograph and were recorded on bromide paper. The amplifier characteristic may be judged by the square wave time signals applied before the filmters at one second intervals (see figures).

’ National Paraplegia Foundation Fellow in Neurology. ‘The term “spontaneous” as applied to fibrillary activity wi.11 be used conventionally in this paper as practically synonymous with “resting”. The accuracy of this convention is questioned in the discussion.

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activity. New units not infrequently appear after the needle has been in place for some time. 1. SUSTAINED ACTIVITY IN DENERVATED MUSCLE A second group of irregularly discharging units can be identified (fig. 1 E). These were Confirming prior workers (Langley and Kato usually active for only a few minutes, sometimes 1914; Brown 1937b; Denny-Brown and Pennyas the terminal activity of regular units of the backer 1938) fairly well sustained fibrillation usufirst type. ally (:an be demonstrated by the fourth or fifth The third group is least common and is day after nerve section, and with increasing ease usually associated with mechanical or severe elecfor the next week or two. However, the large trical stimulation (Kugelberg and Petersen 1949). volumes of tissue which may be electrically silent However, such activity has been seen to recur as or very quiet at this time and even much later, long as thirty minutes after undisturbed placement after considelable atrophy has occurred, are imof the electrode. The patterns are highly similar pressive. That the needle is not situated in inexto those of myotonic muscle. They consist of incitable tissue can be readily shown by the large termittent high frequency bursts (as high as 100 envelope of activity during d,irect current stimulaper sec. for short periods) and last sometimes for tion (fig. 1 A) and during and after mechanical several seconds, usually at a gradually decrementstimulation (fig. 1 B. C and D ) . The best susing frequency (figs. 1 F, G and 4 K, L, N ) . tained "spontaneous" activity is usually observed Smaller irregularly occurring bursts of only a few with the electrode tip at the periphery of the muspotentials may appear in intervals (fig. 1 H ) . A cle just beneath the skin. few irregularly recurring single potentials someConsiderable variety in the pattern of activity times are associated, often with a gradual build-up to the burst (fig. 1 F). In very high frequency in fibrillation was seen but there is no correlation of patterns with length of time after nerve section. bursts (fig. i F, G, H) the reduced size of the The most common type is the regularly repetitive second potential indicates a rate of activity limited potential reported by many authors. Frequencies by the relative refractory period (Adrian 1930). ranging from 30 per sec. to 1 in several seconds This is the only circumstance in which we have have been observed. The faster frequencies are observed significant spontaneous variation in ammost commonly associated with mechan,ical stimu- plitude of a single fibre potential. Apparent varialation, and whenever seen, are not maintained so tions as in figure 4 L (also fig. 2 A ) are evidently long as activity at the lower frequencies. Occasion- due to different degrees of fibre potential synchroally a gradual slowing of frequency occurs. Sin- nization. gle "skipped beats" without change in the overall The gross similarity of these observations to rhythm are seen spon:taneously and during polarthose of Adrian (1930) on the potentials of inization (figs. 1 M, 5 A and E). Longer silent jured mammalian nerve and to those of Adrian intervals of inconstant duration are often seen, and Gelfan (1933) on injured frog muscle is evident. usually just prior to complete cessation of a unit's RESULTS

Fig. 1 In this and subsequent figures the time interval after denervation is indicated for each record. A. 22 days. End of polarization at signal, 15 v(ylts, cathode at ankle. Note quiet of resting record.-B. 8 days. Light percussiofl of muscle belly with finger tip in first strip. Next strip 10 sec. later shows in.creased activi:ty, and last 2 min. later, shows gradual return to silent baseline record.--C. 5 days. Muscle belly squeezed lightly beginning with swing of baseline.--D. Same as C. Relaxation of squeeze at beginning of strip.--E. 21 days. Spontaneous irregular fibrillations.--F. 10 days. Grouped activity. Note changes in potential amplRude.--G. Same as F. Beginning of a burst that lasted several seconds.--H. Same as F. A succession of sma}l grouped discharges.--/. 22 days. Polarization, cathode at knee, 3 volts. Make and break at signals.--]. Same as I. Same stimulus, make and break.--K. Same as I with electrodes reversed and same voltage. Make and break. Sustained repetitive activity was not elicited in I and ]. Note the large spike at break in I and ] and repetitively at make in K.--L. 22 days, Polarization, cathode at knee, 221,~ volts. Make ~t signal.-M. Continuation of L after 1 second. Break a.t signal. Note that the sm~ll pmentia'l, lower row of Jots, changes frequency very little. A second fibre, upper row of dots, is ~ctivated by polarization, and a third, middle row, is inhibited along with most of the baseline activity. Square waves in these and other figures are one second time markers. Time for all records in A.

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Fig. 2 A. Through F. Normal muscle. Needle insertion potentials. Note variations in amplitude and duration. A is a continuous record. Note that double pips probably represent synchronized discharge.--G. Normal rabbit gastrocnemius, double and single motor units. Compare wLth F and H . - - H. 8 days. Large fibrillation potentials.--I. Normal muscle. Polarization with nerve intact (Pfluger's tetanus).--J. Same as I. Repetitive activity induced by direct current after curariz~,tion. Note that complex motor unit poten,tials have changed to simpler fibre dimensions.--K. Normal muscle. Polarizatibn with nerve ir~tact.--L. 18 days. Fibrillation. K and L were recorded successively with .the same electrode and identical amplification. I and J and subsequent similar records were made by cor~tinuous rapid sweeps on paper moving at the same speed as in the conventional records. The obliquity of the baseline is evident. Time for A through H i~, A. 5 msec. time line in I and ]. Time for K and L in K.

The shower of activity which appears with initial manipulation of the electrode in denervated muscle always decrements, rapidly at first, over the course of minutes, unit by unit, frequently to complete s.ilence. Often the moment when mechanical stimulation is actually stopped can not be clearly determined from the potential record. Indeed, sometimes, as in figure 1 B, the activity appears to increase for a few seconds af~:er a mechanical disturbance. The distinction between "induced" and "spontaneous" activity must to a considerable extent be arbitrary, since all activity is recorded after needle insertion, and since random entrance and e~it of fibrillation units is often seen very long after placement. Following needle movement in normal muscle a continuous range of amplitudes and durations of repetitive potentials was observed, some of which undoubtedly represent synchronized activity (fig. 2 A-F). Durations have been seen from ll/z to 50 reset, and amplitudes varied by a hundredfold, to a maximum of 20O to 300 ,~V with the electrodes used. Similar activity has occurred in freshly denervated muscle (second to third day), after neuromuscular transmission has disappeared and before "true" fibrillations are evident, and in curarized normal muscle. Rarely these potentials have persisted for sezeral minutes after mechanical stimulation. It was our general impression that the larger "insertion" potentials were more readily obtained in normal than in denerva~ted muscle, presumably because of the absence of fibrosis and unresponsive tissue in the former. It also seemed that "insertion" activity was more difficult to elicit in normal muscle after 15 to 30 rain. faradic tetanization, than in the resting state. This fatigue may relate to the "warming up" of electrically stimulated denervated muscle (Ravin 1940) and to the '~warming up" of myotonia. 3. SYNCHRONIZATION IN DENI-RVATED MUSCLE

Synchronization has been seen in all patterns of fibrillation activity and at all stages after denervation when single potentials could be demonstr~ted. The criteria for considering activity to be synchronized wi.th blind leading: from a small area

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must be quite rigid. Simply large amplitude, as Jasper and Ballem (1949) observed, may indicate only highly local leading from a single fibre. Unusually long durations of potential change may be similarly explained. Moreover, as Bishop and Gilson ( 1927) demonstrated, asymmetrical leading may explain a \-arkty of polyphasic potential sha-

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The usual similar orientation of spikes relative to the electrode (fig. 3) probably indicates that the fibres involved lie parallel and close together. It appears that the potential in the first fibre is conducted from the active locus toward the electrod,e and also in the opposite direction to the point of cross excitation of the second fibre. A potenbial in the second fibre is then propagated back to the electrode. Assuming that the conducted potential (at 5 meters per sec.) of the first fibre fires the hyperirritable second fibre, the masimum total conducted distance indicated by the time intervals between spikes would be of the reasonable order of 100 mm. The occasional dropping out of one potential or the discontinuous change of amplitude of ‘In ‘lpparently single potential, w
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one often cannot be certain that the pattern does not represent double activity from the same fibre. Figure 4 K and L shows a doublet where the second spike is occasionally missing as the frequency decreases. Very rarely when two fibres are .perfectly synchronized the .dropping out of one of ,them accounts for an abrupt decre~e in amplitude (fig. 4 N ) usually but not always associated with a decrease in frequency. Relatively high voltage long lasting potentials (as contrasted with the smallest fibrillation potentials) were observed, which certa,inly must to some extent represen,t synchronized activity due to mutual facilitation in a localized area. Durations as long as 23 to 50 msec. are not uncommon. Our localized leading may explain why we could not record any potentials even approximating the 200 msec. waves reported by Kugelberg and Peeersen (t949). Variations in discharge pattern of synchronized potentials were sometimes recorded. Usually a synchronized unit behaves exactly like a single fibre, appearing, changing frequency, disappearing, spontaneously and in response m direct current stimulation, without significant change in general configuration. Figure 4 C and D shows a doublet whose spike interval was changed after a light tap on ,the m u s d e belly. I.t is unlikely that this could have moved the needle far enough (4

A. 8 days. Doublet.--B, Same as A a few minutes later. Soon after this record was made the activity stopped, the second spike dropping out first.--C. 8 days. Douhlet. The activity sudden'ly disappeared after about 15 minutes.-D. Same locus as C. The doublet was started again by lightly tapping the muscle belly. Note the prolonged interval between spikes together with the stoyeer frequency. --E. Through ]. 108 days, a~er curarization. Syr~chronized pattern comai.ning at least four units. E and F were taken successively. G, H, and I were taken at 5 minute intervals, and ] immediately after I. The moving forward of the third spike together with minor changes in the general configuration are to be noted.--K. 108 days. Grouped activity of a dot~blet. Note that the larger spike is always preceded by the shorter at a fixed interva£ Three times after the end of the burst the smaller poter*tial occurs alone.--L. Same locus as K. Note the oscillations of spike amplitudes as the second spike t~mporally overrides to various degrees the first spike of the ensuing doublet.--M. 64 days. Polyphasic synchronized pattern. Reading downward the records were taken at approximately 5 min. ir,tervals. Note ,that the last spike appears to move forward and invert the phase of the final portion of the pattern.--N. 10 days. Grouped activity. Note the inte~miRent depression of spike amplitude. Time for all slow records in D. 5 msec. time line for each group of fast records.

POTENTIALS IN DENERVATED MUSCLE

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sion of frequency to complete inhibition. A postinhibitory facilitation of mild degree is seen with the larger currents. Figure 5, D, E, F, G and H shows the reverse effect of stimulation with the cathode at the ankle. With increasing currents one sees concomitant increase in frequency. However, with further increase there occur frequent skipped beats (fig. 5, E) slowing of frequency (fig. 5, F) and finally complete inhibition (fig. 5, G). Figure 5, H shows the general extinction of aclJivity with very large currents, which represents, by definition, contracture (Gasser 1930), since the muscle is strongly contracted, x Whether the cessation of propagated activity in a single fibre (fig. 5, F and G) with smaller polarizing currents represents contracture cannot be surely determined, but it appears ~ikely since the gradual decrease in frequency with increased currents could hardly represent an -effect at other than the focus of rhythmic activity, wh.ich focus is finally maintained in a steady state of polarization. Most of the fibres recorded from one locus 4. POLARIZATION behave in a similar fashion. However, exceptions The conclusions that may be dragon from the are almo~ always seen (fig. 5, B and C). Figure following results are somewhat qualified by the 1, L and M shows a striking example of a locus conditions of the experiment. The orientation of where polarization starts one repetitive fibre, stops the active fibres and the posi,tions of active loci in another, and leaves a third practically unaffected. an undefined electrical field are unknown. That It can be seen that polarizaecion does produce genedifferent fibres do not respond in the same manner ra,l patterns of "anodal" inhibition and "post-' should be expected on anatomical and physical ba- anodal" facilitation, and "cathodal" facilitation ses apart from any individual cell differences. It and less difinite "post-cathodal" inhibition. The is to be expected that the propagated potential frequencies of spontaneously active fibres are alterwhich is recorded is the product of complex pola- ed and there is recruitment and inhibition at varization of ,indeterminate polarity, of trigger area rious thresholds of other repetitive units. Gradual increment of current over a period of seconds and of the entire length of conductive fibre. leads to the same degree of activity produced by a The records of polarized muscle for the first steady current of the same terminal i.nten~ity (fig. few days after nerve section (after curare when 5, I and ]). Long term direct current stimulation neuromuscular conduction is still present) usually (several minutes) results in a gradual falling off show poorly sustained irregular potentials that are difficult to define but are not artefact. After about of activity (accommodation?). Some of the rethe fourth day when "spontaneous" fibrillations petitive potentials in our records dropped out withappear, the activity of polarized muscles is also out changing frequency as Kugelberg and Petersen (1949) have observed, but we have frequently fibrillary. The responses of single fibres can be noted a decrease ia frequency before disappearance. readily followed. Poorly synchronized "twitch" responses are Figure 5 A-H shows the effect of a polarizaseen at make and break of the polarizing current tion on a sustained fibrillation. The frequency in the resting state is quite constant apart from the (figs. 1, I-K, and 5, A and D ) . R can frequently residual effects of previous stimulation. (Note resting record in C and H.) The effect of direct 1As Doupe (1943) pointed out, the "contracture" of current wi.th the cathode at the knee at increasing denervated muscle studied by Bremer (1932) consisted voltages is shown in figure 5 A, B and C, depres- largely of propagated fibre contractions.

to 8 mm.) to account for this change in latency without producing more significant change in potential form and amplitude. It appears therefore that the tap produced a new focus or foci of maximum irritability, i.e., that the pacemaker area on the hypersensitive fibre is not rigidly fixed. Further evidence for this contention can be adduced from spontaneous changes which appear over the course of many minutes. Rarely a doublet has been seen in which the second spike dropped out a few beats before the first. Occasionally over the course of several minutes the spikes have moved farther apart before they successively stop (fig. 4 A and B). In other records the spike interval shortens and subsequently returns ~o the original interval. Doublets as in figure 3 I have gradually become perfectly synchronized as in 3 M. Changes in complex units are seen in figure 4 E-I, M. In general it can be said that the more complex the patterns of synchrony the more subject they are to spontaneous variation.

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WILLIAM M. LANDAU and is seen even at threshold current intensity for "twitch". "Spontaneous" repetitive poten~tials may or may not respond in the "twitch". Ravin's observation ( 1 9 4 0 ) that the first "twi,tch" after a rest is p r o l o n g e d compared with the ensuing responses was confirmed but was not studied in detail. A comparison of visual and palpatory cri,teria for " t w i t c h " threshold to threshold of electrically

be demonstrated that the population of fibres resp o n d i n g at ,the make is to some extent different f r o m that respondi,ng at the break. Response of different fascicles can also be shown sometimes by palpation or visual observation ( D o u p e 1 9 4 3 ) . Reversal of current f l o w results in the previous pattern of the make response appearing ~t the break and vice versa. T h e r e is sometimes a differ-

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Fig. 5 A. 18 days. Polarization. Recording electrode 1/3 way from distal to proximal polarizing electrode. A. Cathode at knee, 5 ~ volts, make and break at signals. Note the initial skipped beat and the decreased frequency during polarization.--B. Same as A. 15 volts.--C. Same as A, a continuous record, 45 volts. Note complete abolition of the large potential and persistence of small one as in B. General post-inhibitory facilitation of activity is seen in B and C.---D. Cathode at ankle, 51/'2 volts, make and break at signals. Note increased frequency during polarization. There is no clear cut poststimulation inhibi~ion.--E. Same as D. 15 volts. Note further increase in frequency with occasional missed beats. Original large spike is indicated by dots.--F. Same as D. 221/2 volts. Note decreased irregular frequency. Original spike is indicated by dots. The other large potential is that first ap~aarent in E and also seen in G and H.--G. Same as D. 45 volts. Complete inhibition of original rge spike• Post-stimulatory facili,tation is seen in some fibres at higher voltages.--H. Same as D. 1121~ volts. Make only. Note almost complete abolition of propagated potentials. Amplification is slightly reduced in H and resting activity has not yet completely recovered from previous strong stimulation. Increase of spike heights in E-H may relate to different needle position during contraction as well as to true amplitude change.--/. 108 days, after curarization. End of polarization at signal, 71/2 volts, cathode at k n e e . - ] . Same locus as I. Signal indicates end of gradually increased polarization (12 see.) to same final value as I. Note identical character of polarization activity. Time for all records in A. ence in latency of response between make and break, and increase of current strength shortens the latencies. Repetitive response of single potentials can be identified both during the shower of potentials o f the "twi,tch" and persisting after it,

recorded response shows fair correlation. There is intrinsic variation in the f o r m e r of 10 per cent o r more, and when disagreement occ6rs, the electrical response is usually present at lower current intensity. Since there is gradual increment of sus-

POTENTIALS IN DENERVATFD MUSCLE tamed "spontaneous" fibrillations with increments of current intensity it is difficult to delimit sharply a threshold for repetitive response. However, the voltage required for such responses progressively falls from 20 to 45 volts a few days after nerve section to 11/2 m 51/2 volts two or three weeks later, far below ,threshold of the intact nerve-muscle prepara.tion. In general, the results confirm the conclusion of Pollock el al. (1945a), that a unitary ratio between .thresholds for galvanic "twitch" and "tetanus" indicates maximal degeneration after nerve section. In further confirmation (Pollock e t a / . , 1945b) .it was found that threshold for "'twitch" diminished by 1/2 to 1/3 in ,the course of a few weeks. It should be pointed ou.t that this may be more apparent than real, since threshold local curren, t density in the muscle of dimin, ished mass may not have changed so greatly. 1 5. DRUGS A series of animals were examined before and after curarization from ,the acute stage of denervation to the 108th day. The repetitive response of direct current nerve stimulation (Pfluger's tetanus) is readily distinguishable .through the first day after denervation (fig. 2, I and K ) . Also during this period curarization resulted in a .two to four fold increase in "twitch" threshold and a three to six fold increase, and even beyond the available v o l t a g e - 159 volts, in ",tetanus" threshold. When a repeti.tive response after curare can be seen clearly above .the "noise" with such large currents (which is seldom), ,it has been .noted that the potentials have changed from motor uni,t to fibre dimensions in records from the same spot (fig. 2, I .and 1)- From the second day on, when nerve stimulation no longer excites the muscle, curare produces no significant change in dectrical thresholds or in recorded activ,Ry. As has been previously observed (Rosenblueth and Luco 1937; Eccles 1941), curarization produced no change in "spontaneous" fibril4ations. (On the other hand, Jasper (1947) has reported depression wi.th curare). Synchronized as well as ' Changes in excitability may be related to the diminished individual fibre diameter in a,trophy. Bishop and O'Lehry (1939) have demonstrated that B and C nerve fibres will respond at the same voltage intensity as A fibres, if the stimulating current is maintained long enough, although with shocks of short duration, the conventional excitability differences are seen. They also found almost no accommodation in C fibres.

177

single fibre activity has been readily followed through drug admin,istration. Since curare does block the effect of injected acetylcholine (Brown 1937b), it appears doubtful that acetylcholine has significant relation to "spontaneous" activity. Prostigmine (.5 mgm. i.v.) produced a few seconds of aggravation of fibrillation at the ,time of injection (Rosenblueth and Luco 1937), but a rapid return to baseline activity occurred when there was still very active fasciculation of normal muscles. Lev.ine, Goodfriend and Soskin (1942), with visual observation reported aggravation of fibrillation in the rabbit, and in clinical electromyography a significant effect of Prostigmine has been emphasized (Weddell et al. 1944; Jasper and Ballem 1949). On the other hand, Harvey and Kuffler (1944) found no effect of Prostigmine in man, and Eccles (1941) found no clear effect of eserine in the rabbi.t, confirming Langley and Kato (1914). In the unanesthetized rabbit Feinstein et al. (1945), found a persistent effect for 15 rain. after injection of 2.5 mgm. of Prostigmine. This suggested that anesthetic might be the significant factor in the difference in results. With an intact CNS Prostigmine might produce an effect analogous to Bender's (1938) fright contraction of denervated muscle in the monkey. However, in two rabbits examined after spinal cordsection at C-5 and after recovery from ether anesthesia, a prolonged effect has not been demonstrated even with doses of Prostigmine as large as 2.5 mgm. DISCUSSION The relatively small volume of denervated muscle which main,tains "spontaneous" fibrillation has not previously been emphasized. Th, is observation opposes the view of Langley (1915) and Tower (1939) that atrophy is in part the result of excessive fibrillary activity, for it is difficult to believe that activity of such a small minority of fibres could be effective in accelerating atrophy. It confirms the conclusions of Solandt and Magtadery (1947) an,d Gutmann and G/rttmann (1944). The evanescer~t and slight effect of Prostigmine in our experiments compared with ,the effectiveness of direct current stimulation (fig. 1 A) suggests that the latter may be useful in the clinical situation where it is important to demonstrate fibrillary activity. It is certainly less uncomfortable

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WILLIAM M. LANDAU

than the administration of large doses of Prostigmine. Obviously 4f the mt~scle contracts visibly with small direct currents, there is no need for electrical recording. The observation that fibrillary activity is most prominent near the surface agrees well with the visual observations of many workers. The absence of tension in denervated muscle, in addition to her visual observations and those of Langley and Kato (1914) indicated to Tower (1939) that fibrillary contractions involved only parts of muscle fibres. In view of the present evidence that sustained electrically excited visible contraction is only an exaggeration of a relatively low degree of resting activity, there seems to be no basis for this hypothesis, also opposed by Hayes and Woolsey (1942) , Brown (1937b) and Denny-Brown and Pennybacker (1938). The anatomical origin of fibri,llary activity, at least ~n the case of cross-excitation of fibres is evidently not always fixed. Since there .is no evidence for variation of electrical excitability along the muscle fibre (Kuffler 1943; Kuffler 1945a), whether or not hyperexcitable as a whole, the changes observed in synchronized activity may be at.tributed to slight variation in relative fibre positions or resistance of interstitial tissues. Although it is probable that isolated fibre activity is usually initiated in the endplate region (Hayes and Woolsey 1942) it should 'be emphasized that denervation" increases the excitability of the entire muscle fibre. Kuffler (1943) showed that the non-endplate zone of ,the denervated fibre has an acetylcholine threshold of the same order as the endplate of the normal fibre. That occasional activity should be initiated in any focally s~imu,lated 4ocus is ,therefore to be expected (Eccles 1941). It is clear that synchronization of activity is restricted to neither normal nor denervated muscle. To be sure, synchronization of fibres in a motor unit is maintained by their common excitation by one nerve fibre, but this is not sufficient reason to indict mechanical nerve fibre stimulation for all recorded synchronized activity as Weddell et al. (1944) have done. They suggested (Feinstein et al. 1945) "that the motor unit ,is a morphological entity which retains its identity for a considerable time after denervation, and responds as a whole to mechan,ical stimuD,tion". Wohlfart's pathological studies (1949) indicate that a motor unit is composed of several or

many discrete and separate small groups of muscle fibres. It appears probable that mutual electric,.1 excitation at a stimulated (injured) area produces large synchronized potentials (Adrian 1930), and that the fibres that respond together are anatomically closely rel.ated. That they represent a neuromuscular unit, however, is a tenuous and unnecessary assumption which is further embarrassed by potentials .larger than ordinary motor units. Kugelberg and Petersen (1949) noted that the first phase of synchronized potentials in denervated muscle was usually positive at a monopolar lead, similar to the "positive sharp waves" of shorter duration reported by Jasper and Ballem (1949) in denervated and myotonic muscles. The latters' suggestion that these are "contraction potentials", related to the positive potentials led from whole muscles by Rosenblueth, Willis and Hoagland (1941) is not supported by the studies of Bishop and Gilson (1927) and of Gelfan and Bishop (1932). It appears more likely that these are effectively monophasic leads from inactive (injured) tissue adjacent to an active locus. The similarity to .the monophasic records of. Bishop and Gilson (1927) 'and Katz and Kuffler (1941) is clear. The search for a specific incident cause for fibrillation such as acetylcholine (Denny-Brown and Pennybacker 1938) or potassium ion (Magladery and Solanck 1942) has ,only emphasized that .the significant feature of denervated muscle is not a specific but a general decrease in response threshold to chemical, electrical, mechanical, thermal, and metabolic stimulation (Kuffler 1943; Feinstein et al 1945). Synchronization is then only a special manifestation of hyperexcitabili.ty. It is not clear, however, that fibrillation is truly spon:taneous activity. If so, it is difficult to explain why such a small proportion .of fibres are in sustained activity when the process of degeneration has presumably gone on for the same length of time in all fibres. The patterns of fibrillation response are strikingly similar .to the injury responses described by Adrian (1930) in mammalian nerve and by Adrian and Gelfan (1933) in frog muscle. With high degrees of exci.tation, synchronized activity was seen in both studies. The slow frequencies and long durations of repetitiveness that are common in fibrillation may indicate that the response mechanism of the muscle whose thres-

POTENTIALS IN DENERVATED MUSCLE hold is lowered by denervation differs somewhat from that of muscle whose threshold is acutely lowered by other means. The difficulty of distinguishing qualitatively among the activity, asynchronous and synchronous, occurring 7spon.taneously" and in response to evident injury in denervation and in myotonia, and after injury .in normal muscle must be admitted. In normal muscle there is rapid accommod,ation to mechanical stimulation; insertion activity is clearly response to mechan~ical injury. Denervated muscle, however, is so characterized by low threshold and lack of accommodation that it is impossible to define the point where insertion potentials stop and "spontaneous" potentials begin. It may be that the question of true spontanei.ty is essentially insoluble. Certainly the :trauma incident to ordinary movements of unshidded muscles is sufficien,t 'to incite fibrillary contractions and needle electrodes certain,ly commit sufficient trauma to negate the sign,ificance of the activity they record. However, if muscle is completely shielded from extraneous ,stimttl,ation it will prgbably undergo degeneration and fibrosis to .the exten,t that it is incapable of contraction, for fibrillation is a criterion of prognostically satisfactory denervated muscle (Weddell et al. 1944; Jasper and Ballem 19a9). It may be more accurate to interpret fibrillation as the abi:li,ty to contract after stimu,lation and to infer that contraction per se is the indication that contractile tissue .is in good condition. Since fibrillary activi~ty cannot be identified entirely by its asynchronous nature and since there is legitimate doubt whether it is truly spontaneous it appears val,id m broaden the definition of fibrillation to include all muscle fibre activity not excited by a J~eural mechanism.

The polarization effects indicate ,that repetitive fibrillations are responses to focal steady states of membrane depolarization (injury potentials?) change of which in eittier direction affects the frequency. The phenomenon of regularly skipped beats indicates .that the trigger area (or another trigger fibre) may discharge rhythmically without necessarily producing a propagated potential. Slow build-up of steady potential to a propagated spike was recorded at the mechanically stimulated site by Adrian and Gelfan (1933). Similar changes have been seen by Kuffler with acetylcholine, KCI, and veratrine (Kuffler 1943;

179

Kuffler 1945a; Kuffler 1945b). These changes are not propagated and it is to be expected that none were seen with needle electrodes. No changes in amplitude of poten,tials have been seen that can certainly be distinguished from possible effects of slight electeode movement during contraction (fig. 5, E, F, G). Increased synchronization of potentials during the heigh.tened activi.ty of polarization was also expected. The absence of such effect suggests the assumption that the anatomical fibre co.n~tiguity necessary for such facilitation is present to only a limited degree in muscle. The observations upon "twitch" responses are exactly what should have been predicted from Doupe's (1943) discussion of strength-dura.tion curves, in part based on Rushmn'.s work (1932). His indictment .of conventional studies of electrical excitability is entirely upheld. Differen:t fibre populations are clearly activated with changes of current orientation and intensity, and repetitive response of some fibres forms a significan,t part of the "twitch" response. Although empirical studies of el.ectrical excitability such as those of Pollock, etc. (1946), are of clinical value, their physiol,ogicaal significance can be made more clear. The curarization studies described here indicate clearly that there is no basis for considering neuro-muscular function significant after the first few days following nerve section, and they only confirm well established physiological facts (Heinbecker et al. 1932; Titeca 1935). Pollock et al. (1946), described a period of Degeneration (presumably of nerve) with a discontinuous excitability curve which persisted For abou,t 30 days af,ter nerve section, to be followed by the period of Denervation, a s~eady state in terms M electrical excitability. This terminology is derived from Adrian's work (1916, 1917), and at the time it was done, it was not known that sectioned nerve fails to activate muscle after two to four days. In discussion of the observation that curarized normal muscle responds to faradism, he admitted that some muscle fibre change must occur with denervation (Adrian 1917), but in discussion of disconti,nuity in strength-duration curves, he wrote (Adrian 1916), "....it shows that during recovery the current takes effect upon .two distinct excitable mechanisms with very different time constants. At first sight it might seem possible to explain this on the

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asst~mption that the current affected two groups of muscle fibres in different states of recovery .... However, if this were the case we would expect to find not one discontinui,ty in the curve but several." a The detailed experimental studies of Pollock, etc. (1944), have shown two or even more discontinuities in many records, but Adrian's conclusion has been accepted nevertheless. The obvious experiment of following strength-duration curves of curarized denervated muscle has never been done. Watts, working with single frog muscle fibres (1924), showed no discontinuities in strengthduration curves after denervafion. A slight elevation occurred with large doses of curare before and after denervation. W e would conclude from present information that changes in muscle fibre excitabili,ty are probably continuous after denervation with some degree of individual variation. Discontinuities of the degenera~tion strength-duration curve would therefore be actually artefact in relation to the interpretation of individual fibre thresholds. The time in reinnervation when motor point stimulation begins to excite nerve effectively is not established. SUMMARY Rabbi.t muscle, from the time of denervation to the 108th day after denervation, and normal, has been studied in situ with needle electrodes. The general nature of "insertion" and of "spontaneous" activity and of synchronization of fibre activity are described in detail. It is demonstrated that direct current stimulation may exaggerate or depress fibrillation activity depending upon the orientation of fibres in the electrical field. Repetitive fibre activity in the "twitch" response at make and break is shown. Curare produces no effec~t upon fibrillation activity and Prostigmine only a transient increment. Fibrillation probably represents repetitive response to casual damage i,n low threshold tissue rather than true spontaneous con,traction. Low threshold rather than fibrillation per se 'is ,the significant factor in denervated muscle and direct current stimulation offers a more effective method of demonstra,ting it than observation of random activity. 'He did not distinguish between discontinuous curves of periods of degeneration and of regeneration and assumed that functional nerve is responsible in both cases for response to currents of short duratinn.

It i,s proposed that ,the definition of fibrilla,tlon activity be enlarged to include al,l muscle fibre contraction not excited by a neural mechanism. The relation of these studies to the significance of strength-duration curve discontinuities is discussed. The encouragement and criticism of Dr. G. H. Bishop are gratefully acknowledged. REFERENCES

ADRIAN, E. D

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ADRIAN,E. D. The effects of injury on mammalian nerve •fibres. Proc. Roy. Soc. Biol., 1930, 106: 596-618. ADRIAN, E. D. and BRONK, D. W. Tile discharge of impulses in motc,r nerve fibres. Part If. The frequency of discharge in reflex and voluntary contractions. J. Physiol., 1929, 67: 119-151. ADRIAN, E. D. and GEI.~AN, S. Rhythmic activRy in skeletal muscle fibres. J. Physiol., 1933, 78: 271-287. BENDER, M. B. Fright and drug reactions in denervated facial and ocular muscles of monkeys. Amer. ]. PhysioL, 1938, 121: 609-619. BISHOP, G. H. and GILSON,A. S. Action potentials accompanying the contractile process in skeletal muscle. Amer. J. Physiol., 1927, 82: 478-495. BISHOp, G. H. and O'LEARY,J. L. B and C nerve fibres. Amer. J. Physiol., 1939, 126: p.434.

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BREMER, F. Researches on the contracture of skeletal muscle. J. Physiol., 1932, 76: 65-94. BROWN, G. L. Action potentials of normal mammalian muscle. Effects of acetylcholine and eserine. J. Ph).G(,L, 1937a, 89: 220-237. BROWN, G. L. The actions of acetylcholine on denervated mammalian and frog's muscle. J. Physiol., 1937b, 89: 438-461. DENNY-BRowN, D. and NEVIN, S. The phenomenon of myotonia. Brain. 1941, 64: 1-18. DF.NNV-BRowN, D. and PENNyBaCK~R, J. B. Fibrillation and fasciculation in voluntary muscle. Brain, 1938, 61: 311-334. Douw, J. Studies in denervation. The contractility and excitabilRy of denervated muscle. ]. NeuroL Neuro.~urg. Psychiat.. 1943, 6: 141-153.

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ECCLES, J. C. Changes produced in muscle by nerve degeneration. Med. J. Aust., 1941, 1: 573-575. ERB, W. Die Thomsen'sche krankheit (myotonia congenita). Studien. Leipzig, F.C.W. Vogel, 1886. quoted in Ravin, A. Myotonia. Medicine, 1939, 18: 443503.

]~EINSTEIN, ~., PATTLE, R. E. and WEDDELL, G. Metabolic factors affecting fibrillation in denervated muscle. J. Neurol. Neurosurg. Psychiat., 1945, 8: 1-11. GASSER, H. S. Contractures of skeletal muscle. Physiol. Rev., 1930, I0: 35-109. GELFAN, S. and BISHOP, G. H. Conducted contractures without action potentials in single muscle fibres. Amer. ]. Physiol., 1933, 103: 237-243.

GUTMANN, E. and GUTTMANN, L. The effect of galvanic exercise on denervated and re-innerva.ted muscles in the rabbit. J. Neurol. Neurosurg. Psych., 1944, 7: 7-17. HARVEY, A. M. and KUVFLER, S. W. Synchronization of spontaneous activity in denervated hmnan muscle. Arch. Neurol. Psychiat., Chicago, 1944, 52: 495-497. HAVES, G. J. and WOOLSEY, C. N. The unit of fibrillary activity and the site of origin of fibrillary contractions in denervated striated muscle. Fed. Proc., 1942, 1: 38. HEINBECKER, P., BISHOP, G. H. and O'LEARY, J. L. Nerve degeneration in poliomyelitis. III. Rate of depression and disappearance of components of conducved action currents in severed nerves; correlation with histologic degeneration in groups of fibres responsible for various components. Arch. Neurol. Ps)chiat., Chicago, 1932, 27: i421-1435. JASPE8, H. H. Integration and disintegration of motor units; Unipolar electromyography in neuromuscular diseases. Fed. Proc.. 1947, 6: 137. JASPER, H. and BALLEM, G. Unipotar electromyograms of normal a-d denervated human muscle. ]. Neurophysiol., 1949, 12: 231-244. KATZ, B. and KUFFLER, S. W. Multiple motor innervation of the frog's sartorius. ]. Neurophysiol., 1941, 4: 209-223.

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PETERSEN, I. and KUGELBERG, E. Duration and form of action potential in the normal human muscle. J. Neurol. Neurosurg. Psychiat., 1949, 12: 124-128. POLLOCK, L. J'., GOLSETH, J. G. and ARIEFF, A~. j. Use of discontinuity of strength-duration curves in muscle in diagnosis of peripheral nerve lesions. Surg. Gynec. Obst., 1944, 79: 133-144. POLLOCK, L. J., GOLSETH, J. G. and ARIEFF, A. J'. Galvanic tetanus and galvanic tetanus ratio in elec-" trodiagnosis of peripheral nerve lesions. Surg. Gynec. Obst., 194'5a, 81: 660-666. POLLOCK, L. J., GOLSETH, J. G., ARI~F~, A. J., SHERMAN, I. C., SCHILLER, M. A. and TIGAY, E. L. Reaction of degeneration in electrodiagnosis of experimental peripheral nerve lesions. I~'ar Med,, 194'5b, 7: 275-283. POLLOCK, L. J., GOLSETH, J. G. and AVdEFF, A. J. Response of muscle to electrical stimuli during degeneration, denervation, and regeneration. A. Research herr. ment. Dis. Proc., 1946, 25: 236-257. RAVIN, A. Observations on denervated muscle in relation to myotonia. Amer. ]. Physiol., 1940, 131: 216-227.

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WEDDELL, G., FEINSTEIN, B. and PATTLE, R. E. The electrical activity of voluntary muscle in man under normal and pathological conditions. Brain, 1944, 67: 178-257.

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Re/erence: LANDAU,W. M. Synchronization of potentials and response to direct current stimulation in denervated mammalian muscle. EEG Clin. Neurophysiol., 1951, 3: 169-182.