A histochemical study of lateral longissimus muscle in rat

EXPERIMENTALNEUROLOGY~,497-518

A Histochemical

(1983)

Study of Lateral Longissimus

SUSAN SCHWARTZ-GIBLIN,LORI

ROSELLO,

Muscle in Rat

AND DONALD

W. PFAFF'

The Rockefeller University, New York, New York 10021 Received April 19, 1982; revision received September 13, 1982 Rat axial muscle previously has not been studied hi&chemically. We were interested in determining the fiber composition and fiber distribution in rat lateral longissimus (LL), the large epaxial dorsitlexor muscle active during sexual posturing in the female rat and to determine ifestrogen replacement in ovariectomixed rats would affect the histochemical protile. Staining for ATPase a&r acid preincubation at pH 4.5, pH 4.35, and after alkaline preincubation at pH 9.4 and staining for NADH-TR revealed that rat LL contains the three major types of fibers present in most mammalian hind limbs: fast-twitch glycolytic (FG); fast-twitch oxidative-glycolytic (FOG); and slow-twitch oxidative (So). The muscle contains predominantly FG fibers, So fibers arc segregated supechially from L2-L6 where they comprise from 11 to 18% of the fiber population, and in an oxidative compartment in the medial deep region of L5 where they comprise 62% of all fibers. In the medial deep region of L5 most of the remaining fast fibers also contain oxidative enzyme. Spindles are most highly concentrated in this oxidative region of L5. Estrogen treatment did not affect the relative number, distribution, or diameter of the three muscle fiber types in rat LL. The concentration of So and FOG fibers and spindles local&d in the region of the lumbosacml joint is discussed by contrasting forceful movements (e.g., rump elevation during sexual behavior) with normal postuml regulation.

INTRODUCTION This study was undertaken to elucidate the histochemical profile of the lateral longissimus muscle, the largest of the rat epaxial postural muscles which is also activated by the final common pathway of the lordosis reflexthe estrogen-dependent, stereotyped, sexual posturing of the female rat in Abbreviations: LL, ML-lateral, medial latissimus, EMG-electromyogram; FG-fast-twitch glycolytic, FOG-fast-twitch oxidative glycolytic; SG-slow-twitch oxidative; F-fast. ’ We gratefully acknowledge Dr. Joan I. Morrell for expert and patient guidance with the photomicrography and Gaby Zummer for preparation of the manuscript. This work was sup ported by U.S. Public Health Service grant HD13795. Please address correspondence to Dr. Susan Schwartx-Giblin, Rockefeller University, 1230 York Avenue, New York City, NY 10021. 497 0014-4886/83/020497-22$03.00/O Copyright All ri&u

@ 1983 by Academic FTes% Inc. of rcprodudion in any form mend.

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response to well defined somatosensory stimulation by the male (34, 35). Participation of this muscle in the reflex has been demonstrated by ablation studies (12) and by ongoing chronic electromyogram (EMG) recordings (50). As described by Brink and PfafF (13), the fibers of the lateral longissimus (LL) take origin from the crest and anterior inner border of the ilium as well as from processes of the lumbar and sacral vertebrae, indirectly by means of the lumbosacral aponeurosis. The aponeurosis partially overlies the muscle and also dips into it. The LL fibers travel in a cranial direction and insert mostly onto lumbar vertebrae but some fibers arising from caudal lumbar vertebrae insert onto the proximal parts of the last three ribs. Because the LL is a prominent postural muscle in rat, e.g., active during hind limb standing (personal observations during chronic EMG recording) that also participates in an important estrogen-dependent reproductive reflex, and because the histochemistry of rat axial muscle has not been investigated, we studied its fiber composition, fiber size, and spindle distribution. Using the LL of ovariectomized rats as controls, we then examined the effect of estrogen replacement (in this way preventing hormone fluctuations in the experimental group) and asked whether or not the estradiol-concentrating cells in the dorsal grey matter of the rat spinal cord at LA, L5, and L6, although relatively few in number, and in the dorsal grey matter (38), could exert an indirect neurotrophic influence on the LL (cf. enhancement of polysynaptic responses of motoneurons innervating a muscle involved in the clasp reflex of Xenopus laevis in the sexually active male compared with the castrate (25). Our results provide a distribution pattern of fiber types in the LL which we are using for EMG electrode placement in chronic recordings from the muscle during the performance of the lordosis reflex. METHODS Animals. The data are derived from two groups of rats ovariectomized at about 2.5 months of age: 12 Long-Evans rats and 12 Sprague-Dawley rats. In the first set of experiments, seven Long-Evans rats were treated with subcutaneous implants of estrogen (Progynon pellet, Schering) and studied histochemically 8 to 44 days later. Hormone-treated and control animals were paired for age (4 to 8 months) when examined, but tissue from both sets of animals was not processed simultaneously. Subsequently, to control for variations in pH, time, and staining procedure, the effect of estrogen pretreatment was studied in 12 Sprague-Dawley rats by examining the animals in three sets of two hormone-treated and two untreated rats. Each set of animals was killed at 4 days to 2 months after subcutaneous implants of estrogen (5-mm estradiol-containing Silastic implants). The animals treated

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with estrogen were tested for estrogendependent lordosis behavior just prior to pmdisse&on anesthesia. Four blocks of lateral longissimus (LL) muscle were removed from each animal at two segmental levels, bilaterally, and tissue sections from each of the four animals of a set were histochemically processed together in the same Coplin jar. Half of the frozen blocks were processed within 1 to 2 days, the other half from 7 to 10 days later without apparent effect. Histochemistry. Animals were anesthetized with Chloropent ( 18 mg/ 100 g body weight). In most cases, the mediaI longissimus (ML) was removed to expose the medial edge of the LL and was discarded. Selected blocks of LL the length of a vertebral segment (about 5 mm) from the rostral border of the L2 vertebra to the caudal border of the L5 vertebra were removed with a razor blade and immediately mounted on frozen chucks and covered with dry ice. On occasion, the entire mediolateral mass of epaxial muscle at a particular segmental level was removed for sectioning. Frozen blocks were stored at -20°C for as long as 10 days without apparent effect on enzyme activity. Serial sections 12 pm thick were cut in a cryostat at - 16’C. Sections were stained either the same day or stored at -20°C for as long as 24 h without apparent change in enzymatic activity. Serial sections were incubated 30 to 60 min at pH 9.4 with 2.7 mM adenosine triphosphate (ATP) and stained with CoClz and NH,$ after tissue fixation and preincubation in alkaline buffer according to the method of Guth and Samaha (3 1). Our preparations were preincubated at pH 9.4 for 20 min to demonstrate alkaline-stable myofibrillar ATPase or preincubated 30 min in acid buffer (31) adjusted to pH 4.35 (K acetate < 50 mu) to demonstrate acid-stable myofibrillar ATPase. To differentiate fast-twitch glycolytic (FG) from fast-twitch oxidative-glycolytic (FOG) by their relative acid sensitivity it was necessary to vary the pH from 4.35 to 4.4 to 4.5 (K acetate 5 50 mM) and vary the preincubation times horn 15 to 30 min, respectively [cf. rat hind limb muscles (49)]. In most, but not all cases, 30min preincubation at pH 4.5 (K acetate < 50 mM) would selectively inhibit the ATPase of FOG fibers. Serial sections were also stained for NADH-tetrazolium reductase (NADHTR) according to the method of Scarpelli et al. as modified in Peruse (42) and in some animals with hematoxylin and eosin. The fiber-type nomenclature we used to describe our results was that of Peter and colleagues (43): fast-twitch glycolytic (FG); fast-twitch oxidativeglycolytic (FOG); and, slow-twitch oxidative (SO) which they based on contractile, biochemical, and histochemical properties. Their histochemical criteria were derived from staining reactions for myosin ATPase at pH 9.4 combined with NADH-diaphorase. With our histochemical method (also staining for ATPase after acid preincubation at pH 4.5 and 4.35), we can

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reveal the fourth fiber type FOG(a) in which the ATPase is more acid-stable than in FOG fibers (comparable to that in the FG) but which also shows the reaction product of NADH-TR. Therefore, our method avoids the pitfall described by Nemeth and Pette (40), i.e., ascribing FG (or IIb) classification to all fibers which show ATPase staining at both pH 9.4 and 4.5. Table 1 compares this nomenclature with the others found in the muscle histochemistry literature. Data QuantiJcation. Fiber types were classified by comparing serial sections stained for ATPase after alkaline and acid preincubations and NADHTR. Then the different fibers types in the superficial region of L3 and L5 and in the medial deep region of L5 were counted in tissue sections stained for ATPase after preincubation 30 min at pH 4.5 (see Table 4). Cells were counted at 100X magnification in a loo-box grid 1 mm* placed in an average region superlicially, and in the area of highest concentration of SO fibers in TABLE 1 Equivalent Muscle Fiber Nomenclature” Authors

FG

FOG

Brook and Kaiser, 1970 (14) Stein nd Padykula, 1962 (53) Burke et al., 1973 (16) Dum and Kennedy, 1980 (23)

IIB’ A FF FF

IIA C FR FR

FOW4

so

F(int) FM)

I B S S

’ Definitions are as follows: FG-fast-twitch glycolytic, FOG-fast-twitch oxidative glycolytic, SO-slow-twitch oxidative. For description of FOG(a) see Methods and Table 2. I, IIA, and IIB are defined in rat gastrocnemius based on myosin ATPase reaction. I-inhibited by preincubation below pH 3.9; IIA-inhibition almost complete after preincubation at or below pH 4.5; IIB-inhibited by preincubation at pH 4.3 or lower. * However, Nemeth and Pette (40), assessingboth myosin ATPase activity after preincubation at pH 4.6 and succinic dehydrogenase (SDH) activity, showed in rat extensor hallucis longus, extensor digitorum longus, and soleus that more than 40% of IIB fibers were not equivalent to FG fibers because they demonstrated high SDH activity. [That subset of IIB fibers is equivalent to FOG(a)]. A, B, and C are defined on the basis of the cytochemical distribution of succinic dehydrogenase activity in rat gastrocnemius. A-an open network of small diformazan particles suggesting a relative sparsity of mitochondria; B-a network of small particles arranged in small polygons or concentric rings; C-large spherical particles in a clearly defined although discontinuous rim of heavy subsarcolemma SDH activity. Also collated with size: A > B > C and with fixed ATPase activity at pH 9.4 without preincubation: C > A > B. A fibers are sensitive to chemical fixation. FF, FR, S, and F(int) are defined in cat medial gastrocnemius: FF-fast-contracting fastfatiguing; FR-fast-contracting fatigue-resistant; S-slow-contracting fatigue-resistant. The FF, FR, and S motor units are composed of FG, FOG, and SO muscle fiber types, respectively. F(int) motor units contain muscle fibers which exhibit ATPase properties of FG fibers and oxidative enzymes like those present in FOG fibers.

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the deeper region of L5. Tissue sections with optimal staining were chosen for counting. The numbers in Table 5 were obtained from sections preincubated 15 min at pH 4.35. For estimation of fiber size, we used the method of “lesser fiber diameter” of Dubowitz and Brooke (22) in which one measures the largest of the smaller diameters of an elliptical or rectangular fiber in cross section; the assumption is that it will reflect the least distortion from the true diameter. In five rats we measured 100 or 150 randomly selected cells of each fiber type. Cells were magnified 450X in sections chosen for quality of stain after preincubation 30 min at pH 4.5. The anatomic distribution of spindles in sections from L3 in 11 muscIes from nine animals and in sections from L5 in 12 muscles from eight of the same animals (estrogen-treated and controls) were obtained as follows. Spindles in one section of tissue from each selected block were confirmed in serial sections for a distance of 100 to 200 pm and then plotted, using a microscopeslide projector, onto an enlarged tracing of that section. Tracings were “squared-off” and the center of each determined, spindle sites were then transferred to one of two composite plots, the center of which corresponded to that of sections of either L3 or L5. The Wilcoxon matched-pairs, signedranks test (two-tailed) was used to statistically compare (i) the number of spindles in the medial half of each section with that in the lateral half of the same section, and (ii) the number of spindles in the medial half of sections from L5 with that in the medial and lateral halves of sections from L3 in the same animal. The number and location of SO fibers in one composite representative section from L3 and one from L5 were obtained by superimposing camera lucida drawings of four sections preincubated 30 min at pH 4.35 from each of those vertebral levels in four different animals. RESULTS Lateral Longissimus Fiber Types. After preincubation at pH 9.4, sections of rat LL from vertebral levels L2 to L6 showed a predominance of darkly staining fibers containing alkaline-stable ATPase. From correlations of myofibrillar ATPase histochemistry and physiologic contraction time of individual motor units in hind limb muscle of cat (16,23,37), and of whole muscle or muscle regions of limb muscles in guinea pig (4), rabbit (43), and lizard (27), and of back muscles in cat (18, 19), we presume that these fibers are fast-twitch fibers and that the LL in rat, as in cat ( 18, 19), is predominantly a fast-contracting muscle. The alkaline-stable fibers form two subsets, as is seen in the section taken from the superficial aspect of L5 (Fig. 1A). The more numerous are large fibers having a mean diameter of 70 f 15.1 pm;

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the others are smaller fibers with a mean diameter of 44 f 9.0 pm. The smaller stain more intensely than the larger after pretreatment with formaldehyde followed by alkaline preincubation as they do in rat tibialis anterior (30, 49, 55). Futhermore, in serial sections using NADH-TR as a marker for oxidation in the tissue, (Fig. lC), the huge fibers showed only a light scattering of formazan granules and were therefore designated FG. The smaller and formaldehyde-resistant fibers showed dense accumulations of formazan granules which in many instances were concentrated in the subsarcolemma region [cf (53)]; these fibers were designated FOG. In the ventral two-thirds of the LL, FG and FOG fibers account for most of the muscle fibers with the exception of segregated medial deep regions at vertebral level L5, to be described below. In most cases in the superficial aspect of LL, FOG fibers were less resistant to acid than were FG fibers. Figure 1B shows blanched FOG fibers indicative of ATPase inhibition following preincubation at pH 4.5, and intermediate staining FG fibers. However, examination of the three serial sections (Fig. 1) revealed that not all FOG fibers were equally acid-sensitive; the arrow points to a formaldehyde-resistant fiber which contained oxidative enzyme but was as acid-resistant as FG fibers. Such fibers were designated FOG(a). They tended to be larger than the other FOG fibers in the superticial part of the muscle where they were infrequently seen. They were most frequent in the deepest one-third of the muscle where they were the only type of oxidative fiber present. The profile of FOG(a) fibers was very similar to that of FR(a) fibers described in cat tibialis anterior (23), but such fibers were not described in rat tibialis anterior or soleus (49). After acid preincubation at pH 4.5 to 4.35, small darkly staining acidstable, (alkaline-labile) ATPase~ontaining fibers (Fig. lA, B) were found segregated predominantly in the superhcial one-third of the muscle from L2 to L6 (see also Fig. 3). They were often seen in association with FOG fibers. In the supertlcial region at L3 they comprised 18% of muscle fibers and had a mean diameter of 39 f 7.6 pm. Serial sections stained for NADH-TR (Fig. 1C) showed these fibers to contain diEuse but dense concentrations of forFIG.1. Serial sections from the super&&l region of the lateral longissimus (LL) at LS. Tissue stained for ATPase after preincubation. A-at pH 9.4, B-at pH 4.5. C-tissue stained for NADH-TR. In A the two blanched fibers in the center of the section are SO, the large intermediate staining fibers are FG (e.g., the four large cells adjacent to the two SO): the most intensely staining fibers are FOG. In B the arrow points to an intiquent fiber type, FOG(a), which stains intensely at pH 9.4, but contains acid-resistant ATF%e at pH 4.5 unlike the other FOG fibers which are blanched in B. In C, note the subsarcolemma distribution of formazan granules in FOG fibers. See Tables 1 and 2. Magnification, X375. Vacuolation in B is freezing artifact.

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mazan granules. From the histochemical-physiologic correlations cited above, we surmised that these were slow-twitch fibers and designated them SO, for their high concentration of NADH-TR. Table 2 summarizes the staining reactions for ATPase and NADH-TR in the different fibers seen in tissue sections from rat LL. Most SO fibers in the LL were found in tissue sections from L5 (Table 3), where, in addition to their superficial distribution, they were concentrated deeper in the muscle along the medial border (Figs. 2, 3B). Here they lay adjacent to a connective tissue septum which penetrated the muscle, coursing medially from its superficial lateral origin. As seen in Fig. 2, in the juxtaseptum area, SO fibers were larger than they were superficially, having a mean diameter of 57 f 13.7 I.crn and constituted 62% of the fiber population. The remaining fibers in the juxtaseptum region (38%) had a mean fiber diameter of 61 + 13.0 pm and acid-labile ATPase (Fig. 2B). Although not seen in the section illustrated in Fig. 2B, which was preincubated at pH 4.5, some of these showed resistance to acidity above pH 4.35. Almost all acidlabile fibers showed formazan granules indicative of the presence of oxidate enzyme (see also Fig. 6). Because their histochemical profile was predominantly that of FOG fibers (cf. Table 2) but their average diameter tended to be greater than that of FOG fibers in the superficial region (cf. Table 4), we designated them F fibers without further specification in the medial deep region of L5. Figure 3A and B illustrates two composite camera lucida drawings of sections of L3 and L5, respectively. Each drawing is of four superimposed sections, each from a different animal. Each dot represents four SO fibers identified by acid-stable ATPase after preincubation at pH 4.35. Table 3 gives the number of SO fibers counted in the medial vs. lateral halves of the composite drawings illustrated in Fig. 3. The chi-square test (3 df) showed that SO fibers were not equiprobable in the four halves (P c 0.001). TABLE 2 Histochemical Profile of Fibers Identified in Serial Sections Enzyme

FG

FOf-34

FOG

SO

ATPase preincubation pH 9.4” pH 4.5 pH 4.35

high inter low

high+ inter low

high+ low low

low high high

NADH-TR

low

inter

high

high

’ Fixed tissue.

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TABLE 3 The Mediolateral Distribution of Spindles and Slow-Twitch Oxidative (SO) Fibers in lQt Lateral Longissimus at L3 and LS

L3 L5

Spindles (No. per section)

SO Fibers (No. per section)’

Lateral

Medial

Lateral

Medial

3.1 2.4

3.5 5.49

295 136

145 559

’ Medial L5 > lateral L3 and L5, P < 0.01, Wilcoxon matched-pairs signed-ranks test. Medial L5 > medial L3, P < 0.05. b Distribution of SO fibers differed from random, Chi-square test, 3 df, P < 0.001.

Estrogen. The histochemistry of LL tissue taken from rats with subcutaneous implants of estrogen showed no difference from control muscle with respect to the relative number of each fiber type, their mean fiber diameter, or location in the tissue. Table 4 summarizes the relative number and fiber diameters of FG, FOG, and SO fibers in LL muscles. Counts and measurements were obtained from tissue sections stained for ATPase after 30-min preincubation at pH 4.5. Consequently, FOG(a) fibers were included in the tabulations of FG fibers in the superficial regions of the LL at L3 and L5. This had little effect on the estimation of FG incidence alone, because in the superficial region of the muscle, FOG(a) fibers were much fewer in number than any other fiber type, and FG fibers constituted a large majority (65 to 73%) ofthe population. Spindles. Spindles were frequently seen in sections of rat LL. They were particularly common in the medial deep oxidative region of L5 (see Fii. 2) and were also often associated with connective tissue septa containing neurovascular bundles. Spindles were not systematically followed in serial sea tions, so the precise numbers of bag and chain fibers [differentiated by relative size (47)] per spindle could not be determined. However, each spindle examined had at least one bag 2 fiber according to the criteria of ATPase intrafusal fiber histochemistry (4 l), i.e., the bag 2 fiber was large and stained darkly after acid preincubation (pH 4.5) and intermediate after alkaline preincubation (pH 9.4). Most spindles also showed a bag 1 fiber which stained with intermediate intensity after acid preincubation and lightly following alkaline preincubation. Chain fibers were not abundant in the sections of spindles examined compared with that of cat limb muscles (10) and cat neck muscles (2,47). Typically, spindles had two chain fibers [cf. (44) rat lumbrical, (54) rat extensor digitorum longus], but spindles with three and four chain fibers were also seen. A typical LL spindle showing bag 1 and bag 2 fibers and two chain fibers is illustrated in Fig. 5A and B; the sections were from

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the capsular region and were stained for ATPase after alkaline and acid preincubation, respectively. Pii 5C shows a spindle, also from the capsular region, containing two bags 2 fibers; such spindles were occasionally seen in rat LL. The location of 172 spindles in sections from L3 in 11 muscles and sections from L5 in 12 muscles were plotted with reference to the center of each section (see Methods). The “scattergrams” in Fig. 4 show the distribution of these spindles with respect to the medial and lateral halves of each section; each dot represents one spindle. Table 3 gives the average number of spindles in each half-section obtained by counting all spindles in Fig. 4 and dividing by the number of sections from which they were derived. Using a Wilcoxon test for matched-pairs and signed-ranks, it is shown that in the same animal (nine muscles, eight animals) more spindles were present in the medial half of L5 than in the lateral half of L5 or L3 (P < O.Ol), and more than in the medial half of L3 (P < 0.05). The medial half of L5 corresponds to the region where SO fibers were most highly concentrated (Fig. 3, Table 3). Medial Longissimus. Four blocks of tissue, two from L3 and two from L5 included the adjacent ML along with the LL. The ML is a long, spindleshape muscle extending from lumbar to caudal vertebrae which at L5 lies medial to the LL superlicially and dorsomedial to the deep region of LL. Contraction of the ML deflects the rat’s tail. The two sections illustrated in Fig. 6 show both muscles stained for ATPase after alkaline preincubation (Fig. 6A) and stained for NADH-TR (Fig. 6B). With both staining procedures, the section became divided into thirds. The dark triangle in the upper right (Fig. 6A) is the ML and shows ATPase staining consistent with a predominantly fast muscle; scattered alkaline-labile SO fibers constituted only 10 and 16% of the total fibers at L3 and L5 levels, respectively. By contrast, the lower third of the section shows the medial deep region of the LL where SO fibers were in high concentration. The more superficial aspect of LL in the middle third of the section showed FG fibers staining less intensely for ATPase than those in the adjacent ML and clusters of darkly staining FOG fibers. In this region blanched SO fibers had a tendency to aggregate with the FOG fibers. FIG. 2. !&al sections from the medial deep region of the LL at L5. Tissue stained for ATPase atIer preincubation. A-at pH 9.4, B-at pH 4.5. In A, SO fibers are blanched and F fibers stain darkly. C-tissue stained for NADH-TR. The arrow points to a spindle containing a bag 1 fiber above (intermediate staining in B), a bag 2 fiber below (darkly staining in B), and a small chain fiber adjacent and to the right of both bag fibers (blanched in B, dark in A, and negative for formazan granules in C). Both bag fibers stain for the presence of NADH-TR in C. Magnification, X375. See Table 2.

(70/l)

Estrogen-treated + SD

37 4.7

39 7.6 37 5.0

44 9.0

L3 + L5 Superhcial

17

11 12 33 16 9 20 12 17 17 22

19 19 16 20 28 16 16 23 17 11

18

FOG

SO

64 8.9

70 15.1

65

70 69 51 64 63 64 72 60 66 67

FG’

11

14 69 8 14 9 14 20 13 6

SO

16

16 13 14 14 10 27 18 13 18

FOG

Mean fiber diameter“ (pm)

(1144/5)

(1001/5)

N

L5 Supcrfrcial

’ Numbers obtained from sections stained for ATPase atIer preincubation at pH 4.5. b No. cells/no. animals. ’ Includes FOG(a) fibers (cf. text). d Method of “lesser fiber diameter” (22).

(350/5)

(1036/5)

(99615)

Controls f SD

Mean (%)

Estrogen-treated

Controls

Nb

L3 Sup&t&al

Relative number (%)

73

70 81 78 78 72 81 59 62 74 76

FG

Relative Number and Mean Diameter of Muscle Fibers in Rat Lateral Longissimus”

TABLE 4

(‘WI

62

59 65 73 62 41 64 78 70 53 56

so

63 9.9

57 13.7

L5 Medial deep (200/5)

(1057/5)

(1027/5)

N

L5 Medial Deep

61 15.3

61 13.0

38

41 35 27 38 59 36 22 30 47 44

F

7

8

“E

8 g

ii 3 b @ “2

x

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A

B

Fro. 3. Composite camera lucida drawings. A-four superimposed cross sections of L3 from four different muscles. B-four superimposed cross sections of L5 from four different muscles. Each dot represents four SO fibers identified by acid-stable ATPase after preincubations at pH 4.35. d, dorsal; m, medial, enlarged approximately X5.

Staining for the presence of NADH-TR (Fig. 6B) showed an interface between the two muscles consisting of nonoxidatiye FG fibers within the ML. As predicted, NADH-TR staining in LL SO and FOG fibers revealed by ATPase However, FOG fibers were preincubation but could be of formazan granules. Table 5 summarizes the relative number and fiber diameters of FG, FOG, and SO fibers in two ML muscles. The counts and measurements were obtained from tissue sections stained for ATPase after 15-min acid preincubation at pH 4.35. ‘-, DISCUSSION Histochemistry revealed that the largest of the rat epaxial muscles, the lumbar dorsiflexor lateral longissimus, contains the three major fiber types present in most hind limb muscles of mammals (1) and that muscle fibers presumed to be fast-twitch based on ATPase and NADH-TR reactions con-

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d f * . . . :: . .* ’ .. :... .* . . . ... .* *. .* . . . . .... . I.: .... . . .

. . ..

. a

Ls

.

. . . .. . .. :..I * ... . . . . -..

.

l

m

.

FIG. 4. The distribution of spindles in individual tissue sections from 11 muscles summed at L3 (A) and from 12 muscles summed at.L5 (B). Each dot represents the locus of a spindle with reference to the center of each section. d, dorsal, m, medial, enlarged approximately X7.

stitute the majority. However, for rat tibialis anterior (24) three identifiable histochemical fiber types have been reported based on succinic dehydrogenase staining affinity but contraction times were brief for all three fiber types (myofibrillar ATPase’was not examined). The histochemical profile correlated with fatigue resistance. The relative numbers of FG, FOG, and SO fibers in rat LL muscles and their respective diameters are remarkably similar to those reported for cat LL ( 19). As in cat, concentrations of slow fibers are segregated within the muscle. In’rat LL, a medial deep region from L5-L6 contains fascicles with a majority of slow fibers; in cat accumulations of slow fibers are seen centrally extending the length of the muscle from L2 to L7. By contrast with cat, the remaining slow fibers in rat LL are also segregated, these are in fascicles in the superficial third of the muscle along its entire length and are not seen in the deeper half of the muscle. These superficial SO fibers may be among those fibers which originate on the medial anterior ilium and travel cranially, progressively inserting into processes of the lumbar vertebrae (13). The segregation of fascicles containing slow fibers (as defined by alkaline-labile ATPase) in predominantly fast muscles has also been described for flexor

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FIG. 5. Two spindles from the capsular region stained for ATPase. A--after preincubation at pH 9.4, the large bag 2 fiber (uppermost) stained with intermediate intensity compared with the lightly staining bag 1 fiber (below) and the two darkly staining small chain fibers. B-a serial section from the same spindle a&x preincubation at pH 4.4. C-a second spindle after preincubation at pH 4.5 showed an intermediate staining bag 1 fiber wedged between two large darkly staining bag 2 fibers. One lightly staining chain fiber is seen symmetrically opposite the bag 1 fiber. Magnification, X1000.

carpi radialis in the cat which has both a superficial and deep accumulation of SO fibers (28,29), for tibiahs anterior (23) but not medial gastrocnemius (16) or extensor digitorum longus (23) in cat, for cat neck muscles splenius and rectus capitus major (46), and for several lizard hind limb muscles (27, 45). The SO fibers are also segregated in biventer cervicis and complexus, two neck muscles in the cat whose proportion of slow fibers is greater than or equal to, respectively, the proportion of FG fibers [FF in (la)]. Muscles examined histochemically for the presence of oxidative and glycogenolytic enzymes, e.g., succinic dehydrogenase and phosphorylase activity or for glycogen, have been described as having oxidative compartments. Examples of such muscles are the medial gastrocnemius (48, 53), tibialis anterior, and plantaris but not the extensor digitorum longus in rat (54), and hind limb muscles in amphibians and lizards [cf. (45) for discussion of oxidative regions and tonic fibers in muscles of all classes of vertebrates]. Such

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LONGISSIMUS

513

muscles are to be contrasted with purely oxidative muscles such as the soleus in cat (32) and rat (53, 54) and hind limb muscles of slow loris (51) which lack FG fibers entirely. Such oxidative compartments as seen also in the rat LL contain fibers with formazan granule distribution typical of FOG and SO fibers and as in rat LL, FOG fibers in these compartments are larger than they are in other regions of the muscle (29). In many lizard hind limb muscles these oxidative pockets are localized only near the joints and it has been suggested that “maintained contraction of these fibers may serve to strengthen or stabilize the joints” (45). In rat LL, tonic activity in the caudal medial deep accumulation of oxidative FOG and SO fibers may function in postural adjustments of the pelvis with respect to the lumbar spine. In our preparation, rat LL, as well as in cat LL (19), cat neck muscle (47), rat hind limb muscle (54), and forepaw flexor carpi radialis muscle of cat (28), spindles tend to be more frequent in the oxidative compartments although they are also noted in other regions of the muscle, e.g., in the region of tendinous inscriptions (47). In cat flexor carpi radialis, spindles are concentrated in the slow fiber region between the tendons of origin and insertion and are never found in the region containing high concentrations of FG fibers (28). In rat LL, whereas spindles were significantly more concentrated in the slow fiber region in the medial aspect of the muscle from L5-L6, we observed a tendency for spindles to be associated with connective tissue septa containing neurovascular bundles throughout the muscle. Barker and Chin (3) found spindles distributed along the intramuscular distribution of the nerve trunk in hind limb muscles. Richmond and Abrahams (47) from their extensive data on spindle distribution in axial neck muscles suggest that for these axial muscles, high spindle density appears to relate mostly to head movement and that the occipitoscapularis which is primarily a scapula rotator (and shows a uniform distribution of its 55% SO fibers) has the lowest spindle density of the five muscles. In this regard, at the other end of the axis, the relative desity of LL spindles caudally may reflect the variety, amplitude, and precision of movements of the pelvis with respect to the lumbar spine compared with small intervertebral movements along the lumbar spine. FIG. 6. Serial sections from LS showing LL and adjacent ML in upper right third. A-&&d for ATPase after preincubation at pH 9.4. The lower third of the section shows a high concentration of blanched SO fibers in the medial deep region of the LL. The middle third of the section is a transition to the superfxial region of the muscle in which the majority of fibers are intermediate staining FG fibers; clusters of dark FOG and SO fibers are also seen. The ML shows only a scattering of SO fibers. B-stained for NADH-TR. The ML contains mostly FG fibers. The lower third shows the oxidative medial deep region of the LL. The subsarcolemma distribution of formazan granules is seen in FOG fibers throughout the LL. Magnification, X42.

(3~2)

41 9.6

39 8.3

Mean fiber diameter (pm)b L3 + L5

11

118 11

9 12 13

10

11 13 11

10 10 10

G+w 1)

50 11.9

79

83 17 77

80 77 80

FG (2703/2)

N

’ Numbers obtained from sections stained for ATPase after 15-min preincubation at pH 4.35. b Method of “lesser fiber diameter” (22).

Controls &SD

Mean (a)

Controls

FOG

so

N

L3

Relative number (96)

16

18

66

84

85 88 82

82 78

15 12 18

69 65

F

88

13 21

18 14

62 71 62

FG

18 22 12

20 15 21

FOG

18 14 17

so

L5

Relative Number and Mean Diameter of Muscle Fibers in Rat Medial Longissimus”

TABLE 5

z

5

gg F “0

.E

8

5

I2 *

ul z

HISTOCHFMISTRY

OF RAT LATFXAL

LONGISSIMUS

515

Slow motor units are the first to be recruited by Ia spindle aBerent fibers [see, e.g., (15, 17,26,32)], and ti tonically owing tdtheir recruitment order and oxidative capacity (32). Because Ia afferent fibers contribute larger monosynaptic excitatory postsynaptic potentials to motoneurons in the same nerve branch of some axial (11) as well as some hind limb [(8, 36); cf. (39)] but not all hind limb muscles (9) and evidence for reflex localization has been reported for both axial (5) and hind limb muscles [(9, 21); cf. (39)], the LL with its multisegmental innervation (13) and compartmentalization of slow fibers and spindles, may provide another example of a muscle whose anatomic organization makes it well suited for segmental proprioceptive reflex control of fine “vernier” movements related to postural adjustments [cf. (6, 7)]. It is highly relevant that a stretch reflex resulting from single or graded pulls on the iliac crest in cat can be recorded only from the central slow fiber region of the LL where spindles are most frequent (20). However, large and forceful ballistic movements such as occur during the lordosis reflex would require the recruitment of fast motor units with large tetanic tensions but would not necessarily require precise control. As rump elevation is contingent upon male stimulation of the skin on the flanks followed by tail base and perineum involving relatively low-threshold pressure receptors (35), it is particularly relevant that in intact cats, rapid ankle extension evoked by low-threshold cutaneous tierent fibers during paw shakes, involves preferential recruitment of the predominantly fast gastrocnemius rather than the soleus muscle (52). Furthermore, electrical stimulation of the sural nerve innervating the skin overlying these muscles will also preferentially recruit the fast motor units in the gastrocnemius (33). REFERENCES 1. ARIANO, M. A., R. B. ARMSTRONG, AND V. R. EDGERTON. 1973. Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21: 51-55. 2. BAKKER, G. J., AND F. J. R. RICHMOND. 1981. Two types of muscle spindlesin cat neck muscles: a histochemical study of intrafusal fibercomposition. J. Neurophysiol. 45: 973986. 3. BARKER, D., AND N. K. CHIN. 1960. The number and distribution of muscle spindles in certain muscles of the cat. J. Anat.94: 473-486. 4. BARNARD, R. J., V. R. EDGERTON, T. FURUKAWA, AND J. B. PETER. 1971. H&chemical, biochemical and contractile properties of red, white and intermediate fibers. Am,J. Physiol.220:410-414. 5. BILCWIQ G., R. H. SCHOR,Y. UCHIN~, AND V. J. WILSON. 1982. Localization of proprioceptive reflexes in the splenius muscle of the cat BrainRes.23%217-221. 6. BINDER, M. D., AND D. G. STUART. 1980. Motor unit-muscle interactions: design features of the neuromuscular control system. Pages 72-98.in Progress in ClinicalNeurophysiology,Vol. 8, SpinalandSupraspinal Mechanisms of VoluntaryMotor ControlandLocomotion.Karger, Basel. 7. ~RMAN, B. R., M. D. BINDER, AND D. G. STUART. 1978. Functional anatomy of the association between motor units and muscle receptors. Am. Zool. 18: 135-152.

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