A distinct difference between slow and fast muscle in acetylcholinesterase recovery after reinnervation in the rat

EXPERIMENTAL

NEUROLOGY

74, 33-50 (1981)

A Distinct Difference between Slow and Fast Muscle in Acetylcholinesterase Recovery after Reinnervation in the Rat WOLF-D.

DETTBARN’

Department of Pharmacology. The Jerry Lewis Neuromuscular Disease Research Center, Vanderbilt University Medical Center. Nashville, Tennessee 37232 Received January 12, 1981; revision received April 4, 1981 The changes in acetylcholinesterase (AChE) and choline acetylttansferase (CAT) activity in nerve proximal and distal to the crush site as well as in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle were studied during denervation and reinnervation in rat. Within 24 h after nerve crush, conduction in the distal nerve and neuromuscular transmission was lost. In the distal nerve segment, AChE and CAT activity showed no initial increase and was reduced to 25% 14 days after crush. During the reinnervation period, AChE and CAT activity recovered to 50% (AChE) and 80% (CAT) of control values and CAT activity in the EDL and SOL muscles followed closely the changes in distal nerve. In muscle, AChE activity was reduced to 15% by 2 weeks postoperatively. Enzyme activity in EDL recovered to 50% of control activity in 5 weeks after crush. In the SOL, end-plate and non-end-plate regions’ AChE activity recovered at a faster rate, resulting in a temporary increase in AChE activity to more than control values during the third and fourth week. By the end of the fifth week, AChE activity had returned to control activity. Turnover values for AChE based on the reinnervation data showed a halflife value for AChE in proximal nerve of 32 days, in distal nerve 42 days, in EDL 23 days, and for SOL 5.1 days. The half-life for AChE in both muscles was significantly shorter than that of the nerve, indicating that the nerve did not supply AChE to the muscles. Half-lives for CAT calculated on the basis of the reinnervation data were I 1.6 days for proximal nerve, 18.4 days for distal nerve, and 30 days for SOL and EDL muscles. It is concluded that the ability to synthesize AChE in endAbbreviations: AChE-acetylcholinesterase, CAT-choline acetyltransferase, SOL-Solens, EDL-extensor digitorum longus. ’ This study was supported by National Institute of Neurological and Communicative Disorders and Stroke grant 1243804, National Institute of Environmental Health Sciences grant 02028-01, and a grant from the Muscular Dystrophy Association of America. The author gratefully thanks Mr. James Conatser and Mrs. Sandra McDaniels for their excellent technical assistance and Mrs. Barbara Page for the typing of the manuscript. 33 0014-4886/8l/lOOO33-18$02.00/0 Copyri@~l 8 1981 by Academic Press. Inc. All rights of reproduction in any form rcscrwd

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plate and non-end-plate regions of muscle is an endogenously programmed event in the development of both fast and slow muscles. The nerve initiates and maintains the synthesis and can modify the rate of synthesis in individual muscle fibers. The mechanism by which the nerve stimulates and maintains AChE synthesis in muscle may be related to the release of trophic factors muscle activity or to a combination of these and other factors still to be investigated.

INTRODUCTION Denervation of muscle results in a variety of morphologic, physiologic,and biochemical changes. Although the mechanisms responsible for those changes are complex and as yet not understood, two factors, trophic agents and muscle activity, seem to play a major controlling role. In previous papers (44, 45), we demonstrated that axotomy of the rat sciatic nerve resulted in a simultaneous decrease in axonal enzymes such as acetylcholinesterase (AChE) and choline acetyltransferase (CAT) as well as AChE in muscle. This finding suggested that innervation was required for the proper maintenance of enzymes closely associated with neuromuscular transmission. The role of AChE and CAT in the hydrolysis and synthesis of acetylcholine (ACh), and their presence in nerve and muscle have been demonstrated (28, 42, 50). After axotomy of rat sciatic nerve, CAT and AChE activity are reduced proximally as well as distally to the injury (34, 57). The loss is greater in the distal segment of the nerve and the denervated muscle (23, 44, 45, 54). Previous investigations of the effects of denervation and reinnervation on AChE and CAT were limited to changes in either the proximal or distal nerve or reinnervated muscle alone (25) and no correlation between these enzymes in regenerating nerve and reinnervated muscle has been established. The simultaneous study of changes in AChE of muscle and nerve during denervation and reinnervation is of great importance because evidence indicates that the enzymes CAT and AChE (43), as well as “trophic factors” (12-14), are moved by axoplasmic transport from the cell body to the axon terminal. Although delivery of AChE or trophic factors across the synapse to the muscle appears to be possible, neither the incorporation of AChE into the postsynaptic site nor the identity of these factors has been established (13, 14, 51). Both muscle activity and the release of neurotrophic substances from nerve appear to be regulatory factors in the control of degradation ( 11, 13, 14) and synthesis of muscle AChE (5, 7, 12, 36, 39, 46, 60). The interpretation of some of these findings remains controversial (6). Other findings indicate that the loss of presynaptic AChE occurs prior to that of the postsynaptic enzyme (44). Thus, in addition to the suggested regulation of muscle AChE synthesis by trophic factors and

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muscle activity, changesin the axonal content and the lossof axonal transport of AChE may also causeloss of AChE at the neuromuscular junction. Preliminary work (8) indicated that the recovery of AChE and CAT activity after nerve crush differed between nerve and muscle as well as between muscle types. Thus, these changes induced by the loss and reestablishment of functional connections between nerve and muscle are a suitable model for studying the mechanismsthat control the functional and metabolic characteristics of muscle. A difference in the rate of loss or recovery of AChE and CAT during denervation and on reinnervation may give an insight into the mechanisms controlling the presence of these enzymes. METHODS Male, Harlan Sprague-Dawley rats ( 160 to 180 g) were anesthetized with ether and the left sciatic nerve was crushed at the sciatic notch over a 3-mm segment for 30 s with a serrated hemostat. Postoperatively, the rats were observed for function of the leg every other day. On pulling the rat gently by the tail on a hard surface, the innervated leg extends and the claws grasp at the surface while the denervated leg drags limply. As the nerve regenerates to the muscle, return to normal function is evident and easily observed. The rats were killed by decapitation at 1, 3, 5, and 7 days and at 2, 3, 4, and 5 weeks after nerve crush. Sciatic nerve, soleus (SOL) and extensor digitorum longus (EDL) muscleswere removed. Prior to removing the nerve, the loss of innervation or the progress of reinnervation was tested by electrical stimulation of the sciatic nerve above and below the site of the crush and the effect on SOL and EDL muscleswas observed. A nerve segment 1 cm in length proximal and a segment 1 cm distal to the crush was removed, cleaned of excesstissue, weighed, and stored on ice. The nerve segments were taken 3 mm above or below the crush and thus were not directly adjacent to the lesion. Control nerve segmentswere taken from similar regions of uncrushed nerves. The SOL and EDL muscles were removed, trimmed of tendons and fat, weighed, and stored on ice. The tissues were minced on a glass plate in ice and then disrupted with a Branson Sonifier-Cell Disrupter 1854 in Ellmann buffer. The tissue concentration was 10 mg/ml. Sonication time for muscle was 1 min with a macroprobe at maximum output. Nerve tissue was sonicated 2 min with a microprobe. Tissues from unoperated animals were used as controls. changes in axotomized nerves and denervated muscles are generally evaluated by comparison with the corresponding counterpart of the experimental animal. Although this approach may eliminate variables introduced by different animals, there is evidence that nerve and muscle con-

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tralateral to a transected sciatic nerve are affected by functional compensation (38, 52). In a few preliminary experiments, rats were perfused 3 -min with 150 ml saline and the AChE and CAT activities of nerve and muscle were compared with those from unperfused animals. Perfusion resulted in no reduction of enzyme activities in agreement with other investigations (55). Unperfused preparations were used throughout the investigation. Acetylcholinesterase Determination. AChE was measured according to the Ellman technique (9). Homogenates were preincubated with tetramonoisopropylpyrophosphortetramide (iso-OMPA 1 X 10V5 M) a specific inhibitor of cholinesterase activity, for 30 min and then assayed with acetylthiocholine (3 X 10e3 M) as substrate. Choline Acetyltransferase Determination. CAT activity was measured according to Fonnum’s technique ( 16). Homogenates were preincubated 15 min with physostigmine sulfate (4 X low4 M) and incubated 2 h at 37°C with a final concentration of 5 X 10d4 M [ 1-14C] acetyl-CoA (specific activity, 1 pCi/pM), 3 X lo-’ M choline iodide, and 2 X 10e4 M physostigmine sulfate. After incubation, samples were placed on ice and lo-p1 samples were removed for extraction and scintillation counting. All measurements were done in triplicate. The activities of AChE and CAT were determined in homogenates and were calculated as nanomoles per milligram per hour, micromoles per muscle per hour, nanomoles per millimeter nerve per hour, and as nanomoles per milligram protein per hour. Enzyme activity expressed in units of whole muscle or length of nerve reflects both changes in weight and in specific activity and is, therefore, more reliable in experiments where manipulations induce changes in weight and protein. Protein was determined by the method of Lowry (37). The data were expressed as a percentage of control and tested (two-tailed) for significance at the 5% level (x0.05). RESULTS After the crush of the sciatic nerve in the midthigh region, transmission to the SOL and EDL muscles was lost within 24 to 48 h. Obvious recovery of leg function was observed between the third and fourth week, and 5 weeks after nerve crush no significant difference from control was apparent. Twitch responses of the SOL and EDL muscles as a result of indirect stimulation above the crush site began to appear between the 12th and 14th day. Muscle twitch height in response to indirect stimulation was 50% of control by the end of the fifth week. Table 1 shows normal values of AChE activity in nerve and muscle. Control proximal nerve samples weighed more and showed higher AChE

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ENZYMES

1

Hydrolysis of Acetylcholine in Nerve and Muscle”

Preparation Proximal nerve Distal nerve Soleus Extensor digitorum longus

nmol ACh/mg tissue/h 66.60 f (15) 41.9 2 (15) 68.62 2 (151 104.92 + (15)

0 Values are the mean +

SE

6.01 3.35 4.00 6.80

nmol ACh/mm nerve/h pmol ACh/ muscle/h 57.91 + 3.51 (1% 38.22 f 2.60 (151 4.93 * 0.49 (15) 10.16 + 0.87 (15)

nmol ACh/mg nrotein h 1188.02 & (15) 719.38 k (151 416.35 -+ (151 725.48 + (15)

101.25 65.32 37.21 26.32

with the number of animals used in parentheses.

activity than the distal nerve segments. This decrease of enzyme in the distal segment may be due either to axons leaving the nerve trunk above the distal segment, or to a decrement of enzyme along the axons themselves. In addition, variable amounts of connective tissue at different levels of the multifascicular nerves such as the sciatic nerve, may lead to an apparent reduction in enzyme activity in the more distal parts of the peripheral nerve. The EDL muscle had a significantly higher AChE activity than the SOL muscle or the sciatic nerve. The CAT activity was significantly higher in nerve than in muscle and no difference was seen between the SOL and EDL muscles (Table 2), which supports the assumption that its location is mainly presynaptic. Denervation and Reinnervation Effects on Weight and Protein. The SOL and EDL muscles followed similar patterns of weight loss. However, the SOL reacted more severely than the EDL to the loss of innervation (Fig. 1). For both muscles, maximum weight loss was seen 14 days after crush. Five weeks after denervation the weights of the SOL and EDL muscles had recovered to 65% of control. Only minor weight changes were observed in the proximal nerve segment, whereas the distal nerve segment showed a significant weight increase within 1 day after injury. Four weeks after the crush its normal weight was restored. Absolute amounts of protein in muscle decreased 20% within 3 weeks after nerve crush. Both muscles regained control values by the end of the 5-week period. Thus, in denervated muscle the total protein content was lost more slowly than the AChE activity (Fig. 2). A greater loss of protein was seen in the distal nerve, with the maximum reduction to 72% 2 weeks

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2

Synthesis of Acetylcholine in Nerve and Muscle”

Preparation

nmol ACh/mg tissue/h

Proximal nerve

nmol ACh/mm nerve/h nmol ACh/ muscle/h

8.64 k 1.21

8.01 + 0.06

(12)

(12)

Distal nerve

3.54 + 0.11

(12)

(12)

Soleus

0.86 k 0.20

100.01 + 6.00

(12)

(12)

1.07 * 0.34

121.12 f 0.42

(12)

(12)

Extensor digitorum longus o Values are the mean +

SE

2.30 f 0.07

nmol ACh/mg protein/h 126.15 f 1.40

(12)

53.21 + 0.70

(12)

7.61 * 1.61

(12) 1.11 + 2.12

(12)

with the number of animals used in parentheses.

after crush. By the end of the fifth week the protein content had recovered to 95% of control. Changes in the proximal nerve were insignificant. Effect of Denervation and Reinnervation on Acetylcholinesterase Activity. Nerve crush was followed by profound changes in the activity of

FIG. 1. Effect of nerve crush on weight of nerve and muscle. Changes in weight of l-cm pieces of proximal (P) and distal (D) nerve stumps and of whole extensor digitorum longus (EDL) and soleus (SOL) muscles. Data are expressed as percentage of control muscles from unoperated animals. Numbers on the ordinate represent percentage of control values. Abscissa represents time in days and weeks after sciatic nerve crush. Broken horizontal line indicates control = 100%. Each point represents mean + SE of at least 10 experiments.

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AChE. Within 3 days,enzyme activity was reduced to about 40% of control in both muscles.Two weeks after nerve crush, AChE activity was reduced to 15% of control in both muscles (Fig. 2). After the secondweek, enzyme activity began to recover in both muscles; however, the rate of recovery in the SOL was much faster than in the EDL. By the end of the fourth week, AChE activity in the SOL had risen to 150% of control whereas the activity in the EDL was only 30% of control. The SOL enzyme activity returned to normal and the EDL had regained only 50% of control activity at the end of the fifth week. The AChE activity in proximal nerve showedan increase to 130%during the initial 24-h period (Fig. 2), followed by a reduction to 50% of control within 14 days after nerve crush and thereafter recovered to 85% of control. In the distal stump, the lossof AChE occurred earlier than in the proximal part of the nerve. No initial increase in enzyme activity was seen. Accumulation of AChE at the distal site of a ligature has been described as rapid, short lasting, and seenonly in a very short segment (5 mm) directly adjacent to a single ligature. In our experiments, no accumulation in the distal segment was seen, possibly because (i) the segment was not taken directly adjacent to the crush site but was removed 3 mm below the crush

FIG. 2. Effect of nerve crush on acetylcholinesterase (AChE) activity. A-changes in AChE activity in proximal (P) and in distal (D) nerves. Enzyme activity is calculated as nanomoles acetylcholine hydrolyzed per millimeter nerve per hour and expressed as percentage of control. B-changes in AChE activity in extensor digitorium longus (EDL) and soleus (SOL) muscles. Enzyme activity is calculated as micromoles acetylcholine hydrolyzed per whole muscle per hour and expressed as percentage of control. Enzyme activity per millimeter nerve or whole muscle reflects both changes in weight and changes in AChE specific activity. See Fig. 1 for further explanation.

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IO' 0135,

OAYS

I

1 3

I 2

WEEKS

AFTER

NERVE

1 4

CRUSH

I 5

0135

DAYS

I

r 2.

WEEKS

I 3

AFTER

NERVE

I 4

I 5

CRUSH

FIG. 3. Effect of nerve crush on acetylcholinesterase (AChE) activity. Changes in AChE activity after nerve crush in proximal (P) and distal (D) nerve segments as well as in EDL and SOL muscles. The results shown are calculated as: A-micromoles acetylcholine (ACh) hydrolyzed per gram muscle per hour; B-micromoles ACh hydrolyzed per gram nerve per hour; C-D-micromoles ACh hydrolyzed per milligram protein per hour and expressed as percentage of control. See Fig. I for further explanation.

site, (ii) it was 1 cm long, and (iii) it was removed 24 h after the crush. Enzyme activity was reduced to 25% 7 days later. The rate of recovery of AChE activity in the distal nerve paralleled that of the proximal nerve; however, by the end of the fifth week enzyme activity was restored only

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to 55% of control. Recovery activity of AChE in nerve and muscle commenced at the same time but proceeded at different rates. In Fig. 3, the time course of changes in AChE activity is shown when calculated on the basis of weight of muscle or nerve and as specific activity. Although the loss of AChE activity correlated well with that described in Fig. 2, there were differences, especially in the recovery rates. In proximal and distal nerve, recovery was near the control values at the end of the fifth week; the same holds for the EDL muscle. The SOL showed a similar rate of rapid recovery as before; however, the increase in activity when calculated on the basis of weight or specific activity was much greater, and remained about double the initial value from the third to the fifth week. The changes of total AChE activity were also studied in end-plate and nonend-plate regions of the SOL. In the SOL, motor end-plates follow a W-shape distribution across the midbelly section at the site of innervation. This region was separated (3 mm on either side of the nerve entry into the muscle) from the relatively end-plate-free regions distal and proximal to this site. No corrections were made for number of end-plates and the data are calculated as micromolar per gram per hour. Because the chosen endplate region also contains muscle fibers, the difference in enzyme activity between end-plate and non-end-plate regions is only an approximation of the true difference. The initial (precrush) AChE activity of the end-plate area was 64.55 + 1.93 PM/g/h and that of the non-end-plate region was 42.27 + 1.26 wM/g/h. As seen from Fig. 4, there was no qualitative difference in the response of AChE activity to crush and reinnervation between end-plate-rich and non-end-plate regions of the SOL. Effects of Nerve Crush and Reinnervation on Choline Acetyltransferase Activity in Nerve and Muscle. The activity of CAT may be regarded as a specific marker for cholinergic nerve terminals in skeletal muscle (54) and thus can be used as a biochemical indicator for the progress of denervation and reinnervation. As seen from Fig. 5, in proximal nerve within 24 h of the crush, there was a rapid increase in CAT activity to 150% of control during the first 3-day period, followed by a reduction to 80% of control at the end of the initial 2 weeks. Thereafter, CAT activity recovered and remained approximately at the precrush level during the remainder of the observation period. Crush of the sciatic nerve was followed by rapid changes of CAT activity in the distal nerve. Within 3 days, the enzyme activity was reduced to less than 30% of control activity. Two weeks after crush, enzyme activity began to increase, finally attaining 80% of control at the end of the fifth week after denervation. The CAT activity changes in the SOL and EDL after the nerve crush were marked but less rapid.

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FIG. 4. Changes in acetylcholinesterase activity after nerve crush in end-plate (EP) and nonend-plate (NEP) regions of soleus muscle. Enzyme activity is calculated as micromoles acetylcholine hydrolyzed per gram muscle per hour and is expressed as percentage of control. See Fig. 1 for further explanation.

Both muscles showed the maximum loss of CAT activity at the end of the 14th day, with the SOL being reduced to 20% and the EDL to 40% of control. The changes in both muscles approximately parallelled those seen in the distal nerve, although CAT activity in the EDL seemed to be less affected by nerve crush. The CAT activity returned at a similar rate in both muscles. By the end of the fifth week, the enzyme activity was still reduced, i.e., 55% of control for the SOL and 80% of control for the EDL. DISCUSSION Crushing the sciatic nerve in the midthigh region caused significant changes in AChE and CAT activity, both in the nerve and in the muscles during the denervation period. AChE activity proximal to the crush in the nerve accumulated rapidly during the initial 24-h period and then decreased. This early increase in AChE activity in the proximal nerve has been shown previously (20,29,43,45,49,58) and is recognized as damming of axonally transported AChE. Enzyme is being transported in excess of removal for at least- 24 h following the crush. The subsequent reduction in AChE activity was less in the proximal than in the distal nerve stump and the rate of loss of AChE activity in the distal nerve was greater in the

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FIG. 5. Effect of nerve crush on choline acetyltransferase (CAT) activity in nerve and muscle. Changes in CAT activity after nerve crush in A-proximal nerves (P) and in distal nerves (D). Enzyme activity is calculated as nanomoles acetylcholine synthesized per millimeter nerve per hour. B-changes in CAT activity in extensor digitorum longus (EDL) and soleus (SOL) muscles. Enzyme activity is calculated as nanomoles acetylcholine synthesized per whole muscle per hour. Data are expressed as percentage of control. See Fig. 1 for further explanation.

first 3 days after crush than during the remaining 1Cday denervation period. Similar qualitative changes were seen for CAT activity; however, the initial accumulation proceeded at a slower rate, presumably due to slower axoplasmic transport of CAT ( 17, 44, 48, 59). The reduction of CAT activity in the proximal segment was smaller (25%) than in the distal nerve segment (75%). After the transient accumulation phase, the subsequent reduction of both enzymes in the proximal segment is due to their decreased synthesis and peripheral transport by the neurons (10, 20, 29, 35). In general, the synthesis and transport of enzymes involved in transmission are decreased during the outgrowth period in favor of materials which are necessary for structural renewal of the axons (3, 49). In the SOL and l?DL muscles, the changes of AChE and CAT activity after nerve crush are similar to those found in the distal nerve during the denervation period. Thus, at 2 weeks after crush, CAT activity was at its lowest in the EDL and SOL with the latter muscle having a significantly . greater loss than the EDL. All animals showed signs of reinnervation within 14 days of the crush, as was judged by determination of increase in muscle weight (Fig. 1), response to electrical stimulation above the crush site, and recovery of the toe-spreading reflex. The rate of growth of the most rapidly growing motor axons is 4.4 mm/day after an initial delay at the crush site of about 2 days

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( 18). In our experiments, the distance between crush site and nerve-muscle contact was 51 mm for the EDL and 48 mm for the SOL. Both muscles responded to indirect stimulation 14 days after crush, indicating a nerve growth rate between 4 and 4.25 mm/day. This is similar to the rate measured for the most rapidly growing sensory fibers (19,40), and agrees well with our data. The CAT activity recovered to nearly control values during reinnervation in both nerve segments, whereas AChE recovery was incomplete. This agrees with the findings that in cut as well as crushed nerves CAT transport recovers faster than that of AChE (43). Other work showed that 4 weeks after nerve crush, the accumulation of CAT and AChE in the regenerating site increased to 130 and to 100% of the contralateral nerve, respectively (20). The bulk of AChE is membrane-bound and only a small fraction is rapidly transported along the axons. AChE of the surface membrane thus arises from local incorporation of intraaxonal enzyme (2). CAT is a soluble enzyme and the different return rates of the activity of these two enzymes seen in regeneration may reflect a different rate of synthesis and a selectivity for transport of these enzymes. From the data in Figs. 2 and 5, assuming first-order kinetics, one can calculate a replacement rate for AChE and CAT in nerve and muscle. Based on the reappearance of CAT activity during the reinnervation period, the half-life for CAT in proximal nerve is 11.67, for distal nerve 18.4, and for SOL and EDL muscles 30 days. Calculations based on the recovery of AChE activity during the reinnervation period indicate half-life values for AChE in proximal nerve 32 days, distal nerve 42 days, EDL 23 days, and SOL 5.1 days. The half-life values of these enzymes in muscle based on their reappearance during the reinnervation period are shorter than those previously reported (58). In that study, the half-lives were 100 days for AChE and 475 days for CAT in the lower hind leg muscles. Those calculations were based on the assumption that the supply of both enzymes to the muscles solely depends on their perikaryonal synthesis and axoplasmic transport. In nerves, the half-life values of AChE and CAT calculated from the reinnervation data agree with the rates established for nerve growth and axoplasmic transport of these enzymes. In both EDL and SOL muscles, however, the half-life for AChE is significantly shorter than in the nerve, thus ruling out the presynaptic synthesis and axoplasmic transport and transfer of AChE to the postsynaptic site as the major source of muscle AChE. The estimates of half-time values for these enzymes, whether based solely on the rate of axonal transport or on recovery of enzyme activity after

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denervation, are probably only approximations of the physiologic rate of turnover of these enzymes and should be understood as such. Nevertheless, they allow certain conclusions. The calculations of turnover rates of 200 days for AChE are based on the assumption that the motor neuron synthesizes, transports, and releases AChE at the neuromuscular junction (58). There is evidence that AChE is transported and released from the nerve terminal (51). No evidence exists, however, that these released molecules are taken up and incorporated into the muscle. Furthermore, only the 4 and 10 S forms are being released, whereas the functionally important 16 S form of AChE is not released from the nerve terminal (51). Additional data suggest that some nerve extract, other than 16 S AChE itself, affects the maintenance of 16 S AChE at the neuromuscular junction (14). The actual measurements of recovery of AChE activity in our experiments indicate quite clearly that the turnover rate for this enzyme is shorter in muscle than in nerve. Furthermore, there are distinct differences in turnover rate when functionally different muscles are investigated such as the EDL and SOL. Both muscles, however, have shorter turnover rates of AChE than the nerve, which makes the suggestion that nerve is the direct and only supplier for muscle AChE highly unlikely. The gradual increase of AChE in EDL during reinnervation may reflect either of two mechanisms. AChE synthesis in a single reinnervated muscle fiber could be a rapid process and the slow increase per whole muscle is then due to the slow addition of newly innervated muscle fibers. Alternatively, the rapid innervation of all muscle fibers followed by slow synthesis of AChE would give similar results (24). According to the experiments reported here, the majority of nerve fibers reached their termination within 2 weeks, at a time when the first electrical and mechanical signs of reinnervation were discovered. Thus, the slow increase probably reflects the rate of synthesis within individual muscle fibers rather than the gradual addition of newly innervated fibers. Although this explanation may hold for the EDL muscle, it cannot explain the rapid recovery and temporary excess of AChE in the SOL during reinnervation. As can be seen from Fig. 4, SOL end-plate as well as nonendplate regions showed a similar rate of loss and recovery of AChE with a temporary increase above control activity. This does not indicate, however, that the specific form of AChE (16 S) which is limited to the end-plate region follows a similar regulation as the 10 S and 4 S forms found in the non-end-plate region of muscle (55). A definite answer, however, has to await a study of the three molecular forms in these two muscle regions during reinnervation. The rapid increase in AChE activity of the SOL during reinnervation

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may be specific for slow muscle and could reflect either of several mechanisms. After nerve crush, the number of innervations to SOL muscle fibers increases (1, 3 1). If some nerve factor regulates the total AChE content in muscle, then such an increase of innervations can explain the increased amount of AChE in the SOL during the early period of reinnervation. With reinnervation accomplished, the muscle fibers will retain only singleaxon innervation and thus enzyme activity returns to normal. Whether or not the increase is caused by the differences in hyperneurotization in these muscles remains to be seen. Earlier investigation into the effects of hyperneurotization on AChE activity failed to demonstrate an increase in AChE activity (25). This earlier study, however, used a different procedure from the present one, such as partial denervation and AChE determination after 1 and 8 days and 16 weeks of denervation. In our experiments, above normal AChE activity was seen in the SOL only in the early reinnervation period 3 to 4 weeks after nerve crush. Alternatively, nerve fibers originally innervating fast muscle (which have higher AChE content) may preferentially reinnervate slow SOL fibers and thus cause a rapid increase in the AChE content. Similar findings were reported for the slow fibers in the deep regions of the gastrocnemius muscle. On random reinnervation of this muscle the slow fibers were converted to fast fibers, while none of the fast fibers in the superficial region of the muscle were converted to slow fiber types (26). The sciatic nerve contains axons supplying different muscle fiber types in many muscles; after denervation, however, axons which innervated fast muscle will not only go to fast muscle but will also innervate slow muscle. Fast-twitch glycolytic fibers have a significantly higher AChE activity than the slow-twitch oxidative fibers of the normally innervated SOL (22). This is also borne out by the data of Table 1. There is no selectivity of muscle fiber types in the reestablishment of innervation (1, 41). In these experiments, most of the slow fibers became reinnervated by fast nerve fibers, due to the numerical preponderance of fast fibers. Furthermore, EDL muscle but not the SOL muscle differentiates between EDL and SOL nerves (32). SOL muscle showed no preference for fibers from either nerve (fast or slow), i.e., its reinnervation is nonselective. The EDL muscle showed a preference for its original nerve fibers over slow or intermediate fibers of the SOL nerve. The third alternative is suggested by previous studies (33) and our own preliminary work. It was observed that nerve crush caused in SOL a significant increase in the type II fibers (fast twitch glycolytic) during the denervation and early reinnervation phase (1 to 4 weeks postcrush). As was suggested, denervated SOL may revert back to a neonatal state when type II fibers dominate and only after full functional innervation do phys-

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