JOURNAL OF NEUROBIOLOGY, VOL. 4, NO. 6, PP. 525-533

SOME PHYSIOLOGICAL AND BIOCHEMICAL FEATURES OF STRIATED MUSCLES REINNERVATED BY PREGANGLIONIC

SYMPATHETIC FIBERS

BEATRIZ RAMIREZ and J. V. LUCO

Department of Neurobiology, Catholic University of Chile, Santiago, Chile

SUMMARY

Skeletal muscle fibers can be reinnervated by motor cholinergic fibers, that is, functional connection can be achieved. However, functional connection implies not only the capacity of the nerve impulse to elicit a contractile response but also the capability of the reinnervating neurons to evoke particular modifications of the physiological and biological features of the muscles.

In order to search for some of the modifications due to reinnervation by preganglionic sympathetic fibers, muscle contraction time was studied in three different preparations of adult cats: a) cricothyroid muscle reinnervated by preganglionic fibers; b) cricothyroideus reinnervated by its own nerve; and c) the corresponding normal neuromuscular prepara- tion. The activities of malic dehydrogenase, of aldolase and pyruvic kinase were studied in these three preparations as well as in the de- nervated cricothyroid muscles.

Reinnervation by preganglionic fibers prolonged the twich contraction time, whereas, self-reinnervation did not alter it. On the other hand, the activities of the three enzymes decreased as a result of denervation. In contrast, the muscle reinnervated with sympathetic preganglionic fibers partially recovered the normal level of malic dehydrogenase and the aldolase activities; but showed no modification in the level of pyruvic kinase activity. Conversely, in the muscle fibers reinnervated by their own nerve, the activity of the three enzymes returned to normal levels. The shortening of contraction time of the preganglionic reinnervated muscle correlates well with the features of the enzymic activities found in these muscles. It can be concluded: a) preganglionic sympathetic axons are able to achieve functional connections with striated muscles and b) considering the trophic effect, preganglionic fibers resemble the motor nerve supplying slow muscles.

INTRODUCTION

Since the classical experiments of Langley and Anderson (1904) it has been known that the preganglionic autonomic cholinergic fibers are able

525 @ 1973 by John Wiley & Sons, Inc.

526 RAMIREZ AND LUCO

to achieve functional connections with skeletal muscles. A functional connection is usually defined as one yielding a contractile response to the stimulation of the reinnervating nerve fibers. Nevertheless, a functional connection may imply other neural influences on the muscle.

It has been demonstrated that some cholinergic motor axons induce the typical contraction of slow muscles, while others provoke the typical contraction of fast muscles (Buller et al., 1960a,b; Eccles, 1967). These effects are related to modifications of the contractile properties of the muscles, and not to changes in elastic properties of the tissue (BuKler and Lewis, 1965). On the other hand, it is known that slow muscle fibers obtain the energy for their contraction mainly from the oxidative breakdown of lipids on their compounds, while fast muscle fibers obtain their energy from anaerobic glycolysis (Dawson and Romanul, 1964; Dubowitz and Pearse 1960; Romanul, 1964). Furthermore, the activities of some key regulatory enzymes for these biochemical processes is neurally regulated (Prewitt and Salafsky, 1967); namely, they depend on the type of cholinergic axons innervating the different muscles (Dubowitz, 1967; Dubowitz and Neuman, 1967; Guth et al., 1968; Prewitt and Salafsky, 1967; Romanul and Van der Meulen, 1966,1967).

The purpose of the present study was to investigate whether the rein- nervation of striated muscle by the cholinergic axons, which normally do not innervate muscle fibers (preganglionic sympathetic fibers), induces a change in their speed of contraction and a correlative modification of some enzymic activities. The contraction time as well as the activity of malic dehydrogenase, aldolase and pyruvic kinase were measured in skeletal muscles reinnervated by preganglionic chloinergic axons of the sympathetic system.

METHODS

Adult cats, anesthetized by an intraperitoneal injection of sodium pentobarbital (Nembutal Abbott 36 mg/kg) , were employed.

Surgical procedure. In a first group of 4 cats the central stump of the cervical sympathetic preganglionic fibers was sutured to the pe- ripheral stump of one of the nerves to the sternothyroid muscle. In a second group of 9 cats the preganglionic fibers were sutured to the external branch of the superior laryngeal nerve which normally innervates the cricothyroid muscle. In a third group of 7 cats the cricothyroideus was denervated. In all three groups the central stumps of the motor nerves were sutured to other muscles of the neighborhood, in order to avoid reinnervation by these nerves (self-reinnervation). In a fourth group of 5 cats the motor nerve innervating the cricothyroideus was sectioned and both stumps were sutured. In all cases the normal contralateral muscle was used as control. Two to four months after surgery physio- logical and biochemical studies were performed.

In order to record the contraction time of the reinnervated cricothyroid muscle and the contralateral normal

Physiological procedure.

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muscle, the cricoid cartilage was transfixed with a thin steel drill and firmly clamped. The trachea was separated from the thyroid cartilage, which was partially isolated, care was taken to preserve the muscle’s blood and nerve supply. In the middle line of the caudal portion of the thyroid cartilage a string was tied and the other end of the string was fixed to a transducer. Care was taken to align the transducer with the direction of the muscle pull. Full precaution was taken to ensure that the muscle contraction approximate as closely as possible to isometric contraction. In order to obtain the maximum twitch tension the muscle’s length was left practically equal to its length a t rest. The transducer was connected to a storage Oscilloscope (Tektronix 564) through a 3C66 Tektronix Carrier amplifier, and the contraction was photographed with a Grass camera. Fibrillary activity was recorded by means of bipolar electrodes. Muscle contraction was evoked by stimulating the cor- responding nerve with an S-4 Grass stimulator. Electrical pulses about 0.5 msec in duration and supramaximal strength were applied. In all the experiments the muscles were covered with mineral oil kept at 37°C.

Enzymic activities were tested in normal cricothyroid muscles, in denervated cricothyroideus, in muscles reinner- vated by preganglionic sympathetic fibers, and in self-reinnervated mus- cles. The muscle tissue was removed, washed in ice-cold water, and homogenized in 0.25M sucrose for 15 sec in an Ultra Turrax (Janke & Junkel K.B.) TP 18/2 homogenizer to prepare a 2% w/v homogenate. The homogenate was centrifuged at 500 g for 30 min and the supernatant used for the enzymic assays. Malic dehydrogenase was determined according to the method described by Ochoa (1955), aldolase according to Baranowski and Niederland (1949) and pyruvic kinase by the method of Bucher and Pfleiderer (1955). Determinations were made spectro- photometrically by measuring the change in absorption a t 340 nm of the nicotinamide adenine nucleotides. The reactions were carried out a t 30°C in 50mM triethanolamine buffer (pH 7.5) with 5mM ethylene- diamine-tetracetate in a final volume of 3 ml. The following reagents (Sigma Chemicals) were used: reduced nicotinamide adenine dinucleotide (NADH), oxaloacetate, fructose diphosphate, d-glycerophosphate de- hydrogenase, adenosine diphosphate, phosphoenol pyruvate, and lactic dehydrogenase. Enzymic activities were expressed as oxidation of NADH in millimoles per minute per gram of wet tissue. It is known that in muscles there is a linear relationship between the weight of the wet tissue and the noncollagenous protein (Godspink et al., 1971).

Biochemical procedures.

RESULTS

A. Contraction Time In preliminary experiments the contractile

response of the sternothyroid muscle was studied a t different frequencies of stimulation in the following preparations: a) normally innervated

Sternothyroid muscle.

528 RAMIREZ AND LUCO

Fig. 1. Response of the sternothyroideus a t the indicated frequencies of stimulation: (a) normal neuromuscular preparation; (b) sternothyroideus reinnervated by its own nerve; (c) muscle reinnervated by preganglionic sympathetic fibers. Calibration time: 10 sec interval.

muscles; b) muscles reinnervated by their own nerves; and c) muscles reinnervated by preganglionic fibers. Responses evoked by stimulating any of the 3 branches, which normally innervate the muscle, were very similar and complete tetanus was obtained between 20-25 shocksl’sec (Fig. la) . Self-reinnervation did not modify these contractile responses; complete tetanus was obtained a t about 20 shocks/sec (Fig. lb ) . How- ever, reinnervation by the preganglionic fibers modifies the whole series of responses (Fig. l c ) ; the complete tetanus was obtained a t abut 10 pulses/ sec. In other words, in the muscle reinnervated by preganglionic fibers, complete tetanus was achieved with about half the frequency needed to tetanize the normal or self-reinnervated muscle.

Cricothyroid muscle. The aforementioned results suggested a more careful study of the effect obtained by reinnervating a skeletal muscle by preganglionic fibers of the sympathetic trunk. The crico- thyroideus was chosen because: aj surgically it was possible to suture the cervical sympathetic nerve with the external branch of the laryngeal nerve, which innervates the muscle; b j it was considered that a small muscle could be more completely reinnervated by the preganglionic fibers; and, c) it was easy to record both the experimental and the control muscles under the same conditions.

I n nine cases a successful reinnervation of the cricothyroid muscle was achieved by suturing the preganglionic trunk to the external branch of

530 RAMIREZ AND LUCO

Table 1 Physiological Features of Cricothyroid Muscles

- Contraction Frequency complete

No of time -. (msec) tetanus ~ (per sec) Series cats X SE X SE __ - ____

A. Normal and own nerve reinnerv. 11 58.5 =t 1.34 18.0 =t 0.84

B. Pre-gangl. reinnerv. 5 105.0 =t 6.32 9 . 4 k 0 . 4 0

P(A - B) 0.001 0.001

a Reinnervation was performed about 3 months before testing. X = mean, SE = standard error, P = significant difference.

The reinnervation by the muscle’s own nerve usually reached a large proportion of the muscle fibers as was shown by the absence of fibrillation or, in some cases, the presence of a minor fibrillary activity. By con- trast, the reinnervation with preganglionic fibers was incomplete, fibrilla- tion was always present in some regions of the muscle. Partial rein- nervation also explains the decrease of tension developed by a twitch. A typical example is given in Figure 2; the twitch tension for the pre- ganglionic muscle preparation was about 45% of the tension developed by the contralateral normal muscle.

B. Enzymic Activity

One oxidative enzyme, malic dehydrogenase, and two glycolytic enzymes, pyruvic kinase and aldolase, were studied since their activities in muscle tissue have been shown to be nerve dependent (Prewitt a.nd Salafsky, 1967). The small amount of tissue available restricted the number of biochemical assays (see Methods) that could be run in each muscle.

Denervation produces a significant decrease of the activities of the three enzymes (Table 2). This decrement was greater for malic de- hydrogenase, confirming other observations (Hogan et al., 1965; Roma- nu1 and Hogan, 1965).

Self-reinnervation of the cricothyroid muscle induced a total recovery of enzymic activities (Table 2), a result that supports observations previously reported (Dubowitz, 1967; Prewitt and Salafsky, 1967; Roma- nu1 and Van der Meulen, 1967).

In muscles reinnervated by preganglionic fibers the activity of pyruvic kinase remained at the value reached after denervation. However, t,he activities of malic dehydrogenase and aldolase increased (Table 2). The increment of the former is highly significant, and the increment of aldolase is in the limit of the degree of significance usually accepted.

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Table 2 Enzymic Activity in Cricothyroid Muscle

(millimoles NADH Oz/min X g wet weight)a ~~

Malic Pyruvic No of dehydrogenase ~ -~ Aldolase ~ kinase

Series cats X SE X SE X SE

A. Normal B. Own nerve

reinnerv. C. Pregangl.

reinnerv. D. Denervated

P(A - B) P(B - C) P(B - D) P(C - D)

20 31.3 f 0.6

5 31.9 f 1 . 9

4 14.8 f 0 . 6 7 4 . 1 f 0.2

0 . 8 0.001 0.001 0.001

5 . 7 f 0 . 4

6.8 f 0 . 9

2 . 3 + 0 . 2 1 .2 f 0 . 1

0.4 0.001 0.001 0.05

10.1 f 0 . 3

9 . 3 f 0 . 6

4 . 1 f 0 . 6 4.7 f 0 . 4

0 . 4 0.001 0.001 0.6

a Reinnervation and ~ denervation were performed about three months before testing enzymic activities. X = mean, SE = standard error, P = significant difference.

DISCUSSION

The time from onset to summit of the muscle twitch, i.e., contraction time, characterizes physiologically slow and fast muscles. The com- parison between two muscles with regard to their contraction time has to be done under standard conditions. The standard adopted by Buller et al. (1960), consisted of the muscle length giving the maximum twitch tension. In the present experiments, care was taken to keep the natural length of the muscles.

Another situation that could disturb such comparison was the presence of nonreinnervated fibers, but this criticism has been ruled out by Buller et al. (1960) , who have demonstrated that following self-reinnervation there is no change in the speed of contraction of the muscles, although the total tension they can achieve is reduced by about 25%. We also observed the maintenance of contraction time in muscles partially rein- nervated by its own nerve.

Therefore, we conclude that the contraction time of striated muscles reinnervated by preganglionic fibers increases by about two times, when compared with the normally innervated muscle. I n other words, pre- ganglionic cholinergic sympathetic fibers are able to establish a functional connection with striated muscles but they are not able to keep their normal speed of contraction, muscle fibers have become slower. The latter effect can be related to different features between the motor cho- linergic axons and the cholinergic axons of the sympathetic preganglionic strand, as follows: a) the amount of transmitter released by the two types of nerves may be different; b) the pattern of impulse traffic may not be the same in both cases; c) the trophic neural influence may be different eithfer qualitatively or quantitatively. The present experi-

532 R A M I R E Z A N D LUCO

ments do not add any data suitable to discuss these possibilities. How- ever, as Buller et al. (1960), pointed out, it seems unlikely that the pattern of impulse traffic could be sufficiently discriminative to account for t,he differentiation of the speed of the muscle contraction.

The fact that the preganglionic reinnervation was incomplete implies that the enzymic measurements were made on a mixture of' both in- nervated and denervated muscle fibers. Nevertheless, any increase in enzymic activities of a partially reinnervated muscle must be located on the innervated fibers.

Considering that the period of reinnervation is preceded by an interval during which the muscle is denervated the influence of the reinnervating nerve should be evaluated by comparing the activity of the enzymes between the denervated and the reinnervated muscle.

Preganglionic fibers that reinnervate a muscle previously denervaited are unable to increase the low pyruvic kinase activity found after denerva- tion. However, these neurons are able to increase malic dehydrogenase as well as aldolase activity. The difference in twitch tension between the normal and the reinnervated muscle suggests that at least about 30-40% of the muscle fibers were reinnervated; a similar proportion corresponds to the increment of malic dehydrogenase and aldolase ac- tivities. The similarity of the two proportions suggests that the malic dehydrogenase and aldolase activity may be fully restored in the rein- nervated muscle fibers. On the other hand, after self-reinnervation, the activity of the three enzymes increases to normal levels.

REFERENCES

BARANOWSKI, T. and NIEDERLAND, T. R. (1949). Aldolase activity of myogen. A.

BUCHER, T. and PFLEIDERER, C. P. (1955). Pyruvate kinase from muscle. In: Methods in Enzymology, Vol. 1, Academic, New Yorlr, 1: 435-436.

BULLER, A. F., ECCLES, J. C. and ECCLES, R. M. (1960a). Differentiation of fast and slow muscles in the cat hind limb.

BULLER, A. J., ECCLES, J. C. and ECCLES, R. M. (1960b). Interactions between motoneurons and muscles in respect of the characteristic speeds of their response. J . Physiol. (London) 150: 417-439.

BULLER, A. J. and LEWIS, D. M. (1965). Further observations on mammalian cross- innervated skeletal muscle.

DAWSON, D. M. and ROMANUL, F. C. A. (1964). Enzymes in muscle 11. Histo- chemical and quantitative studies.

DUBOWITZ, V. (1967). Cross-innervated mammalian skeletal muscle: histochemical, physiological and biochemical observations.

DUBOWITZ, V. and NEUMAN, D. L. (1967). Change in enzyme pattern after cross- innervation of fast and slow skeletal muscle.

DUBOWITZ,, V. and PEARSE, A. G. E. (1960). A comparative histochemical study of oxidative enzyme and phosphorylase activity in skeletal muscle. Histochemie 2:

ECCLES, J. C. (1967). The effects of nerve cross-union on muscle contraction. In: Milhorat, A. T.,

J . Biol. Chem. 180: 543-552.

J . Physiol. (London) 150: 399-416.

J. Physiol. (London) 178: 343-358.

Arch. Neurol. (Chicago) 11: 369-378.

J. Physiol. (London) 193: 481-496.

Nature (London) 214: 840-841.

105-117.

Exploratory Concepts in Muscular Dystrophy and Related Disorders. Ed., Excerpta Medica Foundation, Amsterdam, pp. 151-163.

NEURAL TROPHIC EFFECT 533

GODSPINK, D. F., HARRIS, J. V., PARK, D. C., PARSONS, M. E. and PENNINGTON, Quantitative enzyme studies in denervated extensor digitorum longus

GUTH, L., WATSON, P. K. and BROWN, W. (1968). Effects of cross-reinnervation Exp . Neurol.

HOGAN, E. L., DAWSON, D. M. and ROMANUL, F. C. A. (1965). Enzymatic changes in denervated muscle 11. Biochemical studies. Arch. Neurol. (Chicago) 13: 274-282.

LANGLEY, J. N. and ANDERSON, H. K. (1904). The union of different kinds of nerve fibers. J . Physiol. (London) 31: 365-391.

OCHOA, S. (1955). Malic dehydrogenase from pig heart. In: Methods in Enzy- mology, Vol. 1, Academic, New York, pp. 735-736.

PREWITT, M. A. and SALAFSKY, B. (1967). Effect of cross-innervation on biochemical characteristics of skeletal muscles.

ROMANUL, F. C. A. (1964). Histochemical studies of enzymes in individual muscle fibers.

ROMANUL, F. C. A. and HOGAN, E. L. (1965). Enzymatic changes in denervated muscle I. Histochemical studies. Arch. Neurol. (Chicago) 13: 263-273.

ROMANUL, F. C. A. and VAN DER MEULEN, J. (1966). Reversal of the enzyme pro- files of muscle fibers in fast and slow muscles by cross innervation. Nature (London)

Slow and fast muscles after cross innervation. Enzymatic and physiological changes. Arch. Neurol. (Chicago)

R. J. (1971). and soleus muscles of rats.

on some chemical properties of red and white muscles of rat and cat.

Int . J . Biochem. 2: 427-433.

20: 52-69.

Amer. J . Physiol. 213: 295--300. Enzymes in muscle I.

Arch. Neurol. (Chicago) 11: 355-368.

212: 1369-1370. ROMANUL, F. C. A. and VAN DER MEULEN, J. (1967).

17: 387-402.

This study was supported by grants from the John Simon Guggenheim Memorial Foundation and from The Fondo de Investigacidn, Universidad Cat6lica de Chile.