EXPERIMENTAL
50, 214-225 (1976)
NEUROLOGY
Distribution
of Extrajunctional
Muscle GARY
Dcpwfrr~crtf
of &4vian
of Normal T.
and
PATTERSON
Scicnccs,
Acetylcholinesterase
AND
U~~ivcvsify Reccizrcd
Dystrophic BARRY
147.
oj California,
in
Chickens WILSON
D&s,
1
Califovrlia 95616
J~tly 2-7, 1975
A specific histochemical staining of serial cross sections of frozen muscle samples for AChE activity was performed to investigate the distribution of sarcoplasmic AChE activity and its relationship to the motor end plate in individual muscle fibers of l-, 2-, and 6-week-old normal and dystrophic chickens. A photographic cytophotometric technique was used to determine AChE activity. There were no differences between normal and dystrophic muscle fibers in the distribution or level ‘of AChE activity at both 1 and 2 weeks of age. By 6 weeks, AChE activity had spread to either side of the motor end plates for approximately five times the distance found in normal fibers. In addition, the level of AChE activity had almost tripled in dystrophic fibers in comparison to normal fibers. These findings suggest that dystrophic chicken muscle develops similarly to normal muscle with respect to AChE localization and level of activity for at least 2 weeks following hatching, and then AChE spreads along the muscle fiber from the motor end plate. The data are consistent with the idea that there is a myogenic defect in the maturation of AChE regulation associated with the motor end plate of dystrophic chickens.
INTRODUCTION A number are believed neurons (3, pression of
of physiological and biochemical properties of skeletal muscles to be regulated and maintained by trophic influences of motor 5, 6, 15). In chickens, these trophic influences affect the exmany properties of the muscle enzyme, acetylcholinesterase
( AChE, acetylcholine acetylhydrolase ; E.C.3.1.1.7.). High biochemical activity, three major isozymes, and histochemical sarcoplasmic localization 1 Research reported here was submitted by the first author in partial fulfillment of the M.S. degree in Physiology, June, 1975, University of California, Davis. Supported in part by NIH Grants AM 16716, NS 10957, ES 00202, and the Muscular Dystrophy Association, Inc. The authors gratefully acknowledge the advice and assistance of Dr. T. A. Linkhart, Dr. H. R. Sawyer, Dr. S. Meizel, Dr. R. H. Sawyer, and Ms. P. S. Nieberg. 211 Copyright All rights
@ 1976 by Academic Press. of reproduction in any form
AchE
AND
MOTOR
END
PLATES
215
of AChE occur in embryonic chick muscle (16). In hatched chickens, the AChE activity of fast twitch muscles decreases, only one isozyme is present, and enzyme histochemical activity is localized at the myoneural junction (16, 17). However, the embryonic pattern of AChE is maintained in fast twitch muscles in adult birds with an inherited muscular dystrophy (16). Especially striking is the checkerboard pattern of AChE localization in the sarcoplasm of cross sections of dystrophic muscle fibers, indicating that some fibers are high and some low in AChE histochemical activity outside of the myoneural junction. Limb bud transplant studies (11) have shown that after 34 days of incubation, dystrophic muscle lacks the ability to regulate the AChE in its sarcoplasm and that genetically dystrophic nerves are capable of supporting normal AChE localization in the muscles they innervate. Whether there is any relation between sarcoplasmic AChE activity of dystrophic muscle and the motor end plate itself is the subject of this report. The experiments presented here use enzyme specific histochemical staining of serial cross sections of frozen muscle samples to study the activity of sarcoplasmic AChE in individual muscle fibers of normal and dystrophic chickens and the relationship of sarcoplasmic AChE to the motor end plates in the fibers. MATERIAL
AND
METHODS
Normal chickens (New Hampshire lines 200 and 412) and dystrophic chickens (New Hampshire lines 304 and 413) were maintained at the Department of Avian Sciences, University of California, Davis. The dystrophic lines are characterized by an early onset of muscle hypertrophy of fast twitch muscles such as the biceps muscles. Lines 200 and 304 are inbred lines of chickens (12). Lines 412 and 413 were recently derived from an outcross of dystrophic line 304 chickens. The animals were killed by exsanguination at 1, 2 and 6 weeks ex ovo. The biceps muscles were removed, washed, and stored in cold physiological saline for approximately 60 min. Samples of the tissues were taken from the middle region of the muscles, quick frozen in isopentane/liquid nitrogen, sealed in plastic bags to prevent dehydration, and stored at -20 C for no more than 24 hrs. The frozen samples were serially cross sectioned in an IEC cryostat, model CTD, at a thickness of 10 pm. Muscle samples were sectioned for a distance of at least 0.5 mm. Each section was oriented on a gelatinized glass slide in the same manner as the preceding section. Some samples were also sectioned longitudinally. The sections were air-dried for 30 min, fixed in 4% buffered formalin for 5 min, and rinsed three times for intervals of 5 min in distilled water.
216
PATTERSON
AND
WILSON
The AChE and nonspecific choline&erase (BChE, acylcholine acylhydrolase; E.C.3.1.1.8.) activities were examined by the method of Karnovsky and Roots (8). [In this method, cholinesterases hydrolyze substrates such as acetylthiocholine (ACTC) and butyrylthiocholine (BUTC) resulting in the formation of thiocholine, copper, and ferricyanide metal complexes.] The sections were incubated at pH 6.0 with ACTC and 10m4 &I tetraisopropyl-pyrophosphoramide (iso-OMPA) , a selective inhibitor of nonspecific cholinesterases, with BUTC, or without substrate. The substrates and inhibitor were obtained from Sigma Co., St. Louis, Missouri. AChE activity was operationally defined as the occurrence of stain due to the hydrolysis of ACTC in the presence of iso-OMPA. In order to determine the change in intensity of the stain through time, the sections were incubated in the various media and photographed every 15 min for 2 hr on 35 mm black and white film (Kodak Panatomic X ; Eastman Kodak Co., Rochester, New York) through a Leitz Ortholux microscope at a magnification of 12.5~. Subsequently, sections were routinely incubated for 90 min. All sections in a run were photographed at the same shutter speed, f-stop, and light exposure. All films were routinely developed in Diafine (Acufine, Inc., Chicago, Illinois), similar to a procedure previously described (1). After development, negatives were enlarged five times with a Durst M600 enlarger (Durst Co., Italy). The images of the fibers were focused on a piece of white cardboard [5 x 7 inches] (127.0 x 177.8 mm) placed at the level of the top of the phototube from a Photovolt densitometer, model 520M (Photovolt Corp., New York, New York). A pinhole in the middle of the cardboard allowed light to pass through the negative to the phototube of the densitometer. The pinhole, regardless of the size fiber being examined, exposed only a minute portion of the fiber to the phototube. The lens of the enlarger was set at the same f-stop for all runs, and the film, not the phototube, was moved to ensure there would be no extraneous variations in light transmission. All fibers were read for percentage transmission from areas that had the least amount of AChE activity. RESULTS Frozen sections of muscle were incubated under several conditions to establish the specificity of the stain for AChE, as was done in previous studies (IO, 11, 16-1s). Patterns of staining of cross and longitudinal sections of biceps muscles from 6-week-old normal and dystrophic chickens are shown in Figs. 1 and 2. No stain was detectetl in sections incubated without substrate. The BChE activity (i.e., hydrolysis of BUTC) occurred only at neuromuscular junctions. The AChE activity (i.e., hydrolysis of
AchE
AND
MOTOR
END
PLATES
217
FIG. 1. Sarcoplasmic AChE in cross sections of 6-week muscle: specificity of stain. in normal biceps muscle. (A) ACTC hydrolysis in the presence of lo-’ M iso-OMPA (B) BUTC hydrolysis in normal muscle. (C) Normal muscle incubated without substrate. (D) ACTC hydrolysis in the presence of lo-’ M iso-OMPA in dystrophic biceps muscle. (E) BUTC hydrolysis in dystrophic muscle. (F) Dystrophic muscle incubated without substrate. Abbreviations : m, motor end plate; s, stained fiber; u, unstained fiber. X 110.
ACTC in the presence of iso-OMPA) occurred at neuromuscular junctions and in the sarcoplasmadjacent to them in normal biceps fibers. In addition, fibers from chicks with inherited muscular dystrophy also exhibited AChE in the sarcoplasmoutside of the region of the neuromuscular junctions. Intensity of the AChE stain was determined by subtracting the absorbance of unstained from that of stained fibers in the same section. Such a calculation corrected for absorption of light due to thicknesses of the percentage transslides and the tissues. Unstained fibers averaged 4 x 10m2 mission whether from stained or from unstained sections. Light absorption was constant from section to section. The AChE activity in the samecross section was measured as a function of time to determine the length of the incubation period to be used in the experiments, Staining increased in a linear fashion for at least 90 min (Fig. 3).
218
PATTERSON
AND
WILSON
FIG. 2. Sarcoplasmic AChE in longitudinal sections of b-week muscle: relation to motor end plates. (A) ACTC hydrolysis in the presence of 10e4 M iso-OMPA in normal biceps muscle. (B) BUTC hydrolysis in normal muscle. (C) ACTC hydrolysis in the presence of lo-” iu iso-OMPA in dystrophic muscle. (D) BUTC hydrolysis in dystrophic biceps muscle. Abbreviations : m, motor end plate; s, stained region of the fiber; LI, unstained region of the fiber. X’ote relationship of AChE to the motor end plates. X 110.
The results of examination of AChE at the motor end plate regions of muscles from l-, 2, and 6-week-old chicks are shown in Table 1. The AChE activity in the region of the motor end plate was similar in l- and 2-week-old chicks. However, AChE activity in and around the motor end plate was approximately three times higher in clystrophic compared to normal biceps muscles at 6 weeks of age. In addition, the distance which AChE activity extended from the motor end plate differed between normal and clystrophic muscle. The AChE
AchE
I 15
I 30
AND
MOTOR
I 45
END
, 60
219
PLATES
I 75
I 90
I 105
1
120
TIME MINUTES
FIG. 3. Staining of sarcoplasmic AChE through time. Bach curve represents the staining of an individual fiber from a 6-week-old dystrophic biceps muscle. Each point represents the intensity of the sarcoplasmic stain in the fiber at a particular time during incubation.
activity was restricted to within 45 pm of the motor end plate in normal muscles and extended to more than 150 pm to either side of the motor end plate of dystrophic muscles. Figure 4 depicts the AChE activity of single muscle fibers from normal and dystrophic muscles of 6-week-old chicks. In no case was AChE activity found separated from the neuromuscular junction. In many dystrophic fibers, AChE activity was skewed with regard to the motor end plate, ,extending further along the fiber to one side or to the other of the junction. However, when the individual fibers were averaged together, the compiled curve of AChE activity exhibited remarkable regularity for both normal (Fig. 5) and dystrophic TABLE 1 AChE OF MOTOR END PLATE Age
REGIONS’
Transmission
(week)
Normal 03
1 2 6
1.25 f 0.26 3.50 f 0.93 2.17 i 0.49
Dystrophic (5%) 1.08 f 0.29 3.23 f 0.55 6.13 f 0.54
a Percentage transmission of sarcoplasmic AChE activity at the motor end plate region of biceps muscle fibers of normal and dystrophic chickens at 1, 2, and 6 weeks of age. Average values of four birds, 40 fibers each, age i standard deviations.
220
PATTERSON
AND
NORMAL
WILSON
DYSTROPHIC
642-
0 642-
.
A
A ;
‘\.,
l -..
(
B
,.‘• 1’
i
l\,‘\
0 6C .’4 4L. % 2- ,.-•// \, 0 D 64\ i 2./-( L-.--, 0200 , ,J, , , , , , 200 100
MEP
100
200
DISTANCE MICRONS
FIG. 4. Cytophotometry of AChE centage transmission of sarcoplasmic from biceps muscles of 6-week-old MEP, motor end plate.
in single muscle fibers AChE activity along normal and dystrophic
of 6-week individual chickens.
chickens. Permuscle fibers Abbreviation:
(Fig. 6) muscles. There was no appreciable difference in the distribution of AChE activity of l- and 2-week-old normal and dystrophic chicks (Fig. 7). DISCUSSION The AChE activity of normal chick fast twitch muscles, such as the biceps, pectoral, and posterior latissimus dorsi muscles, is localized to the region of the motor end plate. However, AChE activity is also found in the sarcoplasm of dystrophic fast twitch fibers in cross sections where motor end plates are not present (16, 17). The results of this study demonstrate that this sarcoplasmic AChE activity is continuous with the motor end plates. The findings suggest that dystrophic muscle fibers develop similarly to normal fibers after hatching, with respect to AChE activity, and then lose their ability to regulate the amount and location of the enzyme. The levels of AChE activity and their distribution along the muscle fibers did not differ between l- and 2-week-old normal and dystrophic chicks. By 6 weeks of age, AChE activity of dystrophic fibers was almost three times the normal and had spread to five times the normal distance from the motor end plate.
A&E
I 200
I
AND
MOTOR
I
I
100
END
I MEP
221
PLATES
I
I
100
I
I
200
I
DISTANCE MICRONS
FIG. 5. Distribution of AChE activity in muscle of &week normal chickens. The points are the average percentage transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens. The bars are the standard deviations for each point. Abbreviation: MEP, motor end plate.
The increase and localization of AChE activity in dystrophic muscle support the idea that there is a defect in the neuromuscular junction of the dystrophic chicken (7, 9, 16). Indeed, autoradiography studies show that the AChE content of dystrophic motor end plates is less than normal (7), even though the data presented here indicate it is higher than normal in the sarcoplasm around the motor end plate. Previous studies on AChE activity in embryo muscle in culture (IS), chick embryo plasma (19), and plasma from dystrophic chickens (19) and normal chickens with denervated muscles (10, 16) show that AChE migrates and is released from muscle fibers that are high in activity of this enzyme. The increased level of AChE in dystrophic compared to normal muscle indicates that the difference in localization in the two muscles is due to more than merely diffusion from the motor end plate. One possibility is that dystrophic muscle fibers have more binding sites for AChE outside of the myoneural junctions than normal fibers. There have been few studies of the relative activity of AChE near and at a distance from neuromuscular junctions. Guth, Albers, and Brown (4) found quantitative differences in the cholinesterase activity of extrajunc-
222
PATTERSON
I
I 200
I
I
100
AND
I
I MEP DISTANCE MICRONS
WILSON
I
I
100
I
I 200
I
FIG. 6. Distribution of AChE activity in muscle of 6-\veek dystrophic chickens. The points are the average percent transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens. The bars are the standard deviations for each point. Abbreviation : REP, motor end plate.
tional and junctional regions of rat muscle fibers. In their study, alternate frozen sections were examined histochemically and the sections in between were quantified spectrophotometrically. Unfortunately, the chicken has no muscle region devoid of motor end plates, and their procedure was unsuited to the present study. In the present study, although a plurality of fibers exhibited an equal amount of AChE on each side of the motor end plate, many of the fibers showed more AChE on one side of the motor end plate than on the other side. There is little likelihood that this was a procedural artifact, since the serial cross sections were arranged in the same direction on the glass slides in every run. The cause of this distribution of AChE in single fibers is not known. Perhaps it is related to the direction in which the nerve approaches the muscle fiber. The spread of AChE activity along the muscle fibers of dystrophic chickens is reminiscent of the spread of acetylcholine receptors (AChR) in other systems. In mammals (15) and in birds (9), neural activity has been shown to be associated with localization of AChR and acetylcholine sensitivity at the myoneural junction. When neural activity is interrupted in mammalian muscles, such as occurs following denervation, both AChR
AchE
AND
MOTOR
END
1 WEEK 010 NORYAL
2
'1
223
PLATES 1 WEEK OLD OVSTROPHIC
1
I
0i
. t
40
t .
YEP
40
*0
DISTANCE MICRONS
FIG. 7. Distribution of AChE activity in muscles of I- and Z-week-old chickens. The points are the average percentage of transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens at each age from normal and dystrophic lines. The bars are the standard deviations for each point. Abbreviation : MEP, motor end plate.
(13) and acetylcholine sensitivity (2) spread along the surface of the fibers. However, there is no evidence of a change in the distribution of ACbR or acetylcholine sensitivity in dystrophic chickens (9). It is interesting that denervation leads to an increase of both AChE ( 10) and AChR (9) in the chiken, and to decrease of AChE activity (4) and an increase of AChR (2) in the rat. These findings and the results presented here suggest that AChE activity and AChR may be regulated in different ways in adult muscle from different species. The photographic cytophotometric method applied here has not been previously reported for estimating enzyme activity in muscle, and is potentially of use for other enzyme studies of single fibers. A similar method is described in detail by Adams (1) to quantify the DNA content of Feulgenstained nuclei in mouse thymus and tumor cells. Some important conditions that must be obtained before the method described here can be applied to enzymes include: The enzyme must be the rate-limiting factor in the staining reaction; the stain must be specific for the enzyme being studied : cross sections of the fibers should stain relatively homogeneously ; and, tissue thickness should remain relatively constant from section to section (14).
224
PATTERSON
Kecent findings muscle fibers from and denervation, of the myoneural plasmic AChE in the
myoneural
AND
WILSON
in this laboratory (20, 21) have shown that human persons with muscle abnormalities, such as dystrophy contain AChE activity localized outside of the region junction. Experiments are in progress to see if sarcosuch
abnormal
human
muscles
is
also
contiguous
with
junction.
REFERENCES 1. :io,zars, I,. R., 1968. A photographic cytophotometric method which avoids distributional error. .4&a Cytol. 12 : 3-8. 2. ,L\SEI.SSON, J., 1959. A study of supersensitivity in denervated mammalian skeletal muscle. J. Physiol. 147 : 178-193. 3. (2.0s;~:. R., 1972. Dynamic properties of mammalian skeletal muscle, Physiol. Rezl. 52 : 129-197. 4. GI.TII, I~., R. W. ALBERS, and W. C. BROWN, 1964. Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates. Exp. Nezlrol. 10 : 236-250. 5. GC.TH, L., 1968. “Trophic” influences of nerve on muscle. Physiol. Rczl. 48: 645-687. 6. HARRIS, A. J., 1974. Inductive functions of the nervous system. Amt. Rev. Physiol. 36: 251-305. 7. JEDRZEJCZYK, J., J. WIECKOWSKI, T. RYMAZEWSIXA, and E. A. BARNARD, 1973. Dystrophic chicken muscle : Altered synaptic acetylcholinesterase. Science 180 : 406-308. 8. KARSOVSKY, hI. J., and I,. ROOTS, 1964. A “direct-coloring” thiocholine method for cholinesterase. J. Histochenz. Cytochcnc. 12 : 219-223. 9. LEBEDA, F. J., J. E. WARNICK, and E. X. ALBUQUERQUE, 1974. Electrical and chemosensitive properties of normal and dystrophic chicken muscle. Expt. Al-clrrol. 43: 2137. 10. LISI
50, 214-225 (1976)
NEUROLOGY
Distribution
of Extrajunctional
Muscle GARY
Dcpwfrr~crtf
of &4vian
of Normal T.
and
PATTERSON
Scicnccs,
Acetylcholinesterase
AND
U~~ivcvsify Reccizrcd
Dystrophic BARRY
147.
oj California,
in
Chickens WILSON
D&s,
1
Califovrlia 95616
J~tly 2-7, 1975
A specific histochemical staining of serial cross sections of frozen muscle samples for AChE activity was performed to investigate the distribution of sarcoplasmic AChE activity and its relationship to the motor end plate in individual muscle fibers of l-, 2-, and 6-week-old normal and dystrophic chickens. A photographic cytophotometric technique was used to determine AChE activity. There were no differences between normal and dystrophic muscle fibers in the distribution or level ‘of AChE activity at both 1 and 2 weeks of age. By 6 weeks, AChE activity had spread to either side of the motor end plates for approximately five times the distance found in normal fibers. In addition, the level of AChE activity had almost tripled in dystrophic fibers in comparison to normal fibers. These findings suggest that dystrophic chicken muscle develops similarly to normal muscle with respect to AChE localization and level of activity for at least 2 weeks following hatching, and then AChE spreads along the muscle fiber from the motor end plate. The data are consistent with the idea that there is a myogenic defect in the maturation of AChE regulation associated with the motor end plate of dystrophic chickens.
INTRODUCTION A number are believed neurons (3, pression of
of physiological and biochemical properties of skeletal muscles to be regulated and maintained by trophic influences of motor 5, 6, 15). In chickens, these trophic influences affect the exmany properties of the muscle enzyme, acetylcholinesterase
( AChE, acetylcholine acetylhydrolase ; E.C.3.1.1.7.). High biochemical activity, three major isozymes, and histochemical sarcoplasmic localization 1 Research reported here was submitted by the first author in partial fulfillment of the M.S. degree in Physiology, June, 1975, University of California, Davis. Supported in part by NIH Grants AM 16716, NS 10957, ES 00202, and the Muscular Dystrophy Association, Inc. The authors gratefully acknowledge the advice and assistance of Dr. T. A. Linkhart, Dr. H. R. Sawyer, Dr. S. Meizel, Dr. R. H. Sawyer, and Ms. P. S. Nieberg. 211 Copyright All rights
@ 1976 by Academic Press. of reproduction in any form
AchE
AND
MOTOR
END
PLATES
215
of AChE occur in embryonic chick muscle (16). In hatched chickens, the AChE activity of fast twitch muscles decreases, only one isozyme is present, and enzyme histochemical activity is localized at the myoneural junction (16, 17). However, the embryonic pattern of AChE is maintained in fast twitch muscles in adult birds with an inherited muscular dystrophy (16). Especially striking is the checkerboard pattern of AChE localization in the sarcoplasm of cross sections of dystrophic muscle fibers, indicating that some fibers are high and some low in AChE histochemical activity outside of the myoneural junction. Limb bud transplant studies (11) have shown that after 34 days of incubation, dystrophic muscle lacks the ability to regulate the AChE in its sarcoplasm and that genetically dystrophic nerves are capable of supporting normal AChE localization in the muscles they innervate. Whether there is any relation between sarcoplasmic AChE activity of dystrophic muscle and the motor end plate itself is the subject of this report. The experiments presented here use enzyme specific histochemical staining of serial cross sections of frozen muscle samples to study the activity of sarcoplasmic AChE in individual muscle fibers of normal and dystrophic chickens and the relationship of sarcoplasmic AChE to the motor end plates in the fibers. MATERIAL
AND
METHODS
Normal chickens (New Hampshire lines 200 and 412) and dystrophic chickens (New Hampshire lines 304 and 413) were maintained at the Department of Avian Sciences, University of California, Davis. The dystrophic lines are characterized by an early onset of muscle hypertrophy of fast twitch muscles such as the biceps muscles. Lines 200 and 304 are inbred lines of chickens (12). Lines 412 and 413 were recently derived from an outcross of dystrophic line 304 chickens. The animals were killed by exsanguination at 1, 2 and 6 weeks ex ovo. The biceps muscles were removed, washed, and stored in cold physiological saline for approximately 60 min. Samples of the tissues were taken from the middle region of the muscles, quick frozen in isopentane/liquid nitrogen, sealed in plastic bags to prevent dehydration, and stored at -20 C for no more than 24 hrs. The frozen samples were serially cross sectioned in an IEC cryostat, model CTD, at a thickness of 10 pm. Muscle samples were sectioned for a distance of at least 0.5 mm. Each section was oriented on a gelatinized glass slide in the same manner as the preceding section. Some samples were also sectioned longitudinally. The sections were air-dried for 30 min, fixed in 4% buffered formalin for 5 min, and rinsed three times for intervals of 5 min in distilled water.
216
PATTERSON
AND
WILSON
The AChE and nonspecific choline&erase (BChE, acylcholine acylhydrolase; E.C.3.1.1.8.) activities were examined by the method of Karnovsky and Roots (8). [In this method, cholinesterases hydrolyze substrates such as acetylthiocholine (ACTC) and butyrylthiocholine (BUTC) resulting in the formation of thiocholine, copper, and ferricyanide metal complexes.] The sections were incubated at pH 6.0 with ACTC and 10m4 &I tetraisopropyl-pyrophosphoramide (iso-OMPA) , a selective inhibitor of nonspecific cholinesterases, with BUTC, or without substrate. The substrates and inhibitor were obtained from Sigma Co., St. Louis, Missouri. AChE activity was operationally defined as the occurrence of stain due to the hydrolysis of ACTC in the presence of iso-OMPA. In order to determine the change in intensity of the stain through time, the sections were incubated in the various media and photographed every 15 min for 2 hr on 35 mm black and white film (Kodak Panatomic X ; Eastman Kodak Co., Rochester, New York) through a Leitz Ortholux microscope at a magnification of 12.5~. Subsequently, sections were routinely incubated for 90 min. All sections in a run were photographed at the same shutter speed, f-stop, and light exposure. All films were routinely developed in Diafine (Acufine, Inc., Chicago, Illinois), similar to a procedure previously described (1). After development, negatives were enlarged five times with a Durst M600 enlarger (Durst Co., Italy). The images of the fibers were focused on a piece of white cardboard [5 x 7 inches] (127.0 x 177.8 mm) placed at the level of the top of the phototube from a Photovolt densitometer, model 520M (Photovolt Corp., New York, New York). A pinhole in the middle of the cardboard allowed light to pass through the negative to the phototube of the densitometer. The pinhole, regardless of the size fiber being examined, exposed only a minute portion of the fiber to the phototube. The lens of the enlarger was set at the same f-stop for all runs, and the film, not the phototube, was moved to ensure there would be no extraneous variations in light transmission. All fibers were read for percentage transmission from areas that had the least amount of AChE activity. RESULTS Frozen sections of muscle were incubated under several conditions to establish the specificity of the stain for AChE, as was done in previous studies (IO, 11, 16-1s). Patterns of staining of cross and longitudinal sections of biceps muscles from 6-week-old normal and dystrophic chickens are shown in Figs. 1 and 2. No stain was detectetl in sections incubated without substrate. The BChE activity (i.e., hydrolysis of BUTC) occurred only at neuromuscular junctions. The AChE activity (i.e., hydrolysis of
AchE
AND
MOTOR
END
PLATES
217
FIG. 1. Sarcoplasmic AChE in cross sections of 6-week muscle: specificity of stain. in normal biceps muscle. (A) ACTC hydrolysis in the presence of lo-’ M iso-OMPA (B) BUTC hydrolysis in normal muscle. (C) Normal muscle incubated without substrate. (D) ACTC hydrolysis in the presence of lo-’ M iso-OMPA in dystrophic biceps muscle. (E) BUTC hydrolysis in dystrophic muscle. (F) Dystrophic muscle incubated without substrate. Abbreviations : m, motor end plate; s, stained fiber; u, unstained fiber. X 110.
ACTC in the presence of iso-OMPA) occurred at neuromuscular junctions and in the sarcoplasmadjacent to them in normal biceps fibers. In addition, fibers from chicks with inherited muscular dystrophy also exhibited AChE in the sarcoplasmoutside of the region of the neuromuscular junctions. Intensity of the AChE stain was determined by subtracting the absorbance of unstained from that of stained fibers in the same section. Such a calculation corrected for absorption of light due to thicknesses of the percentage transslides and the tissues. Unstained fibers averaged 4 x 10m2 mission whether from stained or from unstained sections. Light absorption was constant from section to section. The AChE activity in the samecross section was measured as a function of time to determine the length of the incubation period to be used in the experiments, Staining increased in a linear fashion for at least 90 min (Fig. 3).
218
PATTERSON
AND
WILSON
FIG. 2. Sarcoplasmic AChE in longitudinal sections of b-week muscle: relation to motor end plates. (A) ACTC hydrolysis in the presence of 10e4 M iso-OMPA in normal biceps muscle. (B) BUTC hydrolysis in normal muscle. (C) ACTC hydrolysis in the presence of lo-” iu iso-OMPA in dystrophic muscle. (D) BUTC hydrolysis in dystrophic biceps muscle. Abbreviations : m, motor end plate; s, stained region of the fiber; LI, unstained region of the fiber. X’ote relationship of AChE to the motor end plates. X 110.
The results of examination of AChE at the motor end plate regions of muscles from l-, 2, and 6-week-old chicks are shown in Table 1. The AChE activity in the region of the motor end plate was similar in l- and 2-week-old chicks. However, AChE activity in and around the motor end plate was approximately three times higher in clystrophic compared to normal biceps muscles at 6 weeks of age. In addition, the distance which AChE activity extended from the motor end plate differed between normal and clystrophic muscle. The AChE
AchE
I 15
I 30
AND
MOTOR
I 45
END
, 60
219
PLATES
I 75
I 90
I 105
1
120
TIME MINUTES
FIG. 3. Staining of sarcoplasmic AChE through time. Bach curve represents the staining of an individual fiber from a 6-week-old dystrophic biceps muscle. Each point represents the intensity of the sarcoplasmic stain in the fiber at a particular time during incubation.
activity was restricted to within 45 pm of the motor end plate in normal muscles and extended to more than 150 pm to either side of the motor end plate of dystrophic muscles. Figure 4 depicts the AChE activity of single muscle fibers from normal and dystrophic muscles of 6-week-old chicks. In no case was AChE activity found separated from the neuromuscular junction. In many dystrophic fibers, AChE activity was skewed with regard to the motor end plate, ,extending further along the fiber to one side or to the other of the junction. However, when the individual fibers were averaged together, the compiled curve of AChE activity exhibited remarkable regularity for both normal (Fig. 5) and dystrophic TABLE 1 AChE OF MOTOR END PLATE Age
REGIONS’
Transmission
(week)
Normal 03
1 2 6
1.25 f 0.26 3.50 f 0.93 2.17 i 0.49
Dystrophic (5%) 1.08 f 0.29 3.23 f 0.55 6.13 f 0.54
a Percentage transmission of sarcoplasmic AChE activity at the motor end plate region of biceps muscle fibers of normal and dystrophic chickens at 1, 2, and 6 weeks of age. Average values of four birds, 40 fibers each, age i standard deviations.
220
PATTERSON
AND
NORMAL
WILSON
DYSTROPHIC
642-
0 642-
.
A
A ;
‘\.,
l -..
(
B
,.‘• 1’
i
l\,‘\
0 6C .’4 4L. % 2- ,.-•// \, 0 D 64\ i 2./-( L-.--, 0200 , ,J, , , , , , 200 100
MEP
100
200
DISTANCE MICRONS
FIG. 4. Cytophotometry of AChE centage transmission of sarcoplasmic from biceps muscles of 6-week-old MEP, motor end plate.
in single muscle fibers AChE activity along normal and dystrophic
of 6-week individual chickens.
chickens. Permuscle fibers Abbreviation:
(Fig. 6) muscles. There was no appreciable difference in the distribution of AChE activity of l- and 2-week-old normal and dystrophic chicks (Fig. 7). DISCUSSION The AChE activity of normal chick fast twitch muscles, such as the biceps, pectoral, and posterior latissimus dorsi muscles, is localized to the region of the motor end plate. However, AChE activity is also found in the sarcoplasm of dystrophic fast twitch fibers in cross sections where motor end plates are not present (16, 17). The results of this study demonstrate that this sarcoplasmic AChE activity is continuous with the motor end plates. The findings suggest that dystrophic muscle fibers develop similarly to normal fibers after hatching, with respect to AChE activity, and then lose their ability to regulate the amount and location of the enzyme. The levels of AChE activity and their distribution along the muscle fibers did not differ between l- and 2-week-old normal and dystrophic chicks. By 6 weeks of age, AChE activity of dystrophic fibers was almost three times the normal and had spread to five times the normal distance from the motor end plate.
A&E
I 200
I
AND
MOTOR
I
I
100
END
I MEP
221
PLATES
I
I
100
I
I
200
I
DISTANCE MICRONS
FIG. 5. Distribution of AChE activity in muscle of &week normal chickens. The points are the average percentage transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens. The bars are the standard deviations for each point. Abbreviation: MEP, motor end plate.
The increase and localization of AChE activity in dystrophic muscle support the idea that there is a defect in the neuromuscular junction of the dystrophic chicken (7, 9, 16). Indeed, autoradiography studies show that the AChE content of dystrophic motor end plates is less than normal (7), even though the data presented here indicate it is higher than normal in the sarcoplasm around the motor end plate. Previous studies on AChE activity in embryo muscle in culture (IS), chick embryo plasma (19), and plasma from dystrophic chickens (19) and normal chickens with denervated muscles (10, 16) show that AChE migrates and is released from muscle fibers that are high in activity of this enzyme. The increased level of AChE in dystrophic compared to normal muscle indicates that the difference in localization in the two muscles is due to more than merely diffusion from the motor end plate. One possibility is that dystrophic muscle fibers have more binding sites for AChE outside of the myoneural junctions than normal fibers. There have been few studies of the relative activity of AChE near and at a distance from neuromuscular junctions. Guth, Albers, and Brown (4) found quantitative differences in the cholinesterase activity of extrajunc-
222
PATTERSON
I
I 200
I
I
100
AND
I
I MEP DISTANCE MICRONS
WILSON
I
I
100
I
I 200
I
FIG. 6. Distribution of AChE activity in muscle of 6-\veek dystrophic chickens. The points are the average percent transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens. The bars are the standard deviations for each point. Abbreviation : REP, motor end plate.
tional and junctional regions of rat muscle fibers. In their study, alternate frozen sections were examined histochemically and the sections in between were quantified spectrophotometrically. Unfortunately, the chicken has no muscle region devoid of motor end plates, and their procedure was unsuited to the present study. In the present study, although a plurality of fibers exhibited an equal amount of AChE on each side of the motor end plate, many of the fibers showed more AChE on one side of the motor end plate than on the other side. There is little likelihood that this was a procedural artifact, since the serial cross sections were arranged in the same direction on the glass slides in every run. The cause of this distribution of AChE in single fibers is not known. Perhaps it is related to the direction in which the nerve approaches the muscle fiber. The spread of AChE activity along the muscle fibers of dystrophic chickens is reminiscent of the spread of acetylcholine receptors (AChR) in other systems. In mammals (15) and in birds (9), neural activity has been shown to be associated with localization of AChR and acetylcholine sensitivity at the myoneural junction. When neural activity is interrupted in mammalian muscles, such as occurs following denervation, both AChR
AchE
AND
MOTOR
END
1 WEEK 010 NORYAL
2
'1
223
PLATES 1 WEEK OLD OVSTROPHIC
1
I
0i
. t
40
t .
YEP
40
*0
DISTANCE MICRONS
FIG. 7. Distribution of AChE activity in muscles of I- and Z-week-old chickens. The points are the average percentage of transmission values of sarcoplasmic AChE activity of 40 fibers from biceps muscles of four chickens at each age from normal and dystrophic lines. The bars are the standard deviations for each point. Abbreviation : MEP, motor end plate.
(13) and acetylcholine sensitivity (2) spread along the surface of the fibers. However, there is no evidence of a change in the distribution of ACbR or acetylcholine sensitivity in dystrophic chickens (9). It is interesting that denervation leads to an increase of both AChE ( 10) and AChR (9) in the chiken, and to decrease of AChE activity (4) and an increase of AChR (2) in the rat. These findings and the results presented here suggest that AChE activity and AChR may be regulated in different ways in adult muscle from different species. The photographic cytophotometric method applied here has not been previously reported for estimating enzyme activity in muscle, and is potentially of use for other enzyme studies of single fibers. A similar method is described in detail by Adams (1) to quantify the DNA content of Feulgenstained nuclei in mouse thymus and tumor cells. Some important conditions that must be obtained before the method described here can be applied to enzymes include: The enzyme must be the rate-limiting factor in the staining reaction; the stain must be specific for the enzyme being studied : cross sections of the fibers should stain relatively homogeneously ; and, tissue thickness should remain relatively constant from section to section (14).
224
PATTERSON
Kecent findings muscle fibers from and denervation, of the myoneural plasmic AChE in the
myoneural
AND
WILSON
in this laboratory (20, 21) have shown that human persons with muscle abnormalities, such as dystrophy contain AChE activity localized outside of the region junction. Experiments are in progress to see if sarcosuch
abnormal
human
muscles
is
also
contiguous
with
junction.
REFERENCES 1. :io,zars, I,. R., 1968. A photographic cytophotometric method which avoids distributional error. .4&a Cytol. 12 : 3-8. 2. ,L\SEI.SSON, J., 1959. A study of supersensitivity in denervated mammalian skeletal muscle. J. Physiol. 147 : 178-193. 3. (2.0s;~:. R., 1972. Dynamic properties of mammalian skeletal muscle, Physiol. Rezl. 52 : 129-197. 4. GI.TII, I~., R. W. ALBERS, and W. C. BROWN, 1964. Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates. Exp. Nezlrol. 10 : 236-250. 5. GC.TH, L., 1968. “Trophic” influences of nerve on muscle. Physiol. Rczl. 48: 645-687. 6. HARRIS, A. J., 1974. Inductive functions of the nervous system. Amt. Rev. Physiol. 36: 251-305. 7. JEDRZEJCZYK, J., J. WIECKOWSKI, T. RYMAZEWSIXA, and E. A. BARNARD, 1973. Dystrophic chicken muscle : Altered synaptic acetylcholinesterase. Science 180 : 406-308. 8. KARSOVSKY, hI. J., and I,. ROOTS, 1964. A “direct-coloring” thiocholine method for cholinesterase. J. Histochenz. Cytochcnc. 12 : 219-223. 9. LEBEDA, F. J., J. E. WARNICK, and E. X. ALBUQUERQUE, 1974. Electrical and chemosensitive properties of normal and dystrophic chicken muscle. Expt. Al-clrrol. 43: 2137. 10. LISI
