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Quantitative changes in the frog muscle spindle with passive stretch
© 1971 by Academic Press, Inc. J. U L T R A S T R U C T U R E R E S E A R C H
36, 743-756 (1971)
743
Quantitative Changes in the Frog Muscle Spindle with Passive Stretch ~ ULF L. KARLSSON,2 WILLIAM M. HOOKER,3 and ELIZABETH G. BENDEICH2
Dental Research Laboratory, College of Dentistry, and Department of Anatomy, College of Medicine, University of Iowa, Iowa City 52240 Received December 30, 1970 Ultrastructural changes in the frog intrafusal were qualitatively and quantitatively evaluated with passive stretch. The following observations were made: (1) The reticular zone was stretchable and remained stretched in the stationary phase. (2) Reticulomeres (unisarcomeric muscle processes in the reticular zone) appeared to be series-coupled with the extracellular material. (3) Extracellular filamentous material transformed from a random orientation to a nonstretchable periodic (810 A) pattern (extromeres). (4) The inner capsule sleeve decreased in diameter. (5) Groups of intrafusal nuclei rearranged from a group to a string pattern. (6) Nerve bulbs changed their contours from rounded to elliptoid. (7) Leptomere spacings increased less than sarcomere spacings. The significance of these changes is discussed in terms of spindle function. The muscle spindle responds to stretch with a characteristic discharge pattern that is divided into a dynamic and a static phase (6). The dynamic peak frequency increases with greater initial lengths of the spindle as well as with increased stretch velocity (10). The static phase frequency increases with increasing lengths of the spindle. Other characteristics are an off-effect at cessation of m o t o r stimulation and a decline of the response at maintained m o t o r stimulation (1). The basic mechanism for these discharge patterns is unknown. Mechanical stretch is the stimulus ultimately governing the net electrical response. U n d o u b t e d l y the morphological organization determines this response. The differen1 This investigation was supported by the General Research Support Grant to University of Iowa, Colleges of Dentistry and Medicine and the Neurosensory Center, Program Project Grant Number NSO3354 of the National Institute of Neurological Diseases and Stroke. Neurosensory Center, Publication 221. 2 Present address: Dental Research Laboratory, College of Dentistry, University of Iowa, Iowa City, Iowa 52240. 3 Present address: Department of Anatomy, School of Medicine, Loma Linda University, Loma Linda, California 92354.
744
KARLSSON, HOOKER AND BENDEICH C o m p a c t zone
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FIo. 1. Schematic representation of measurable periodicities in and around the compact and reticular zones of an intrafusal unit in the frog muscle spindle. At the levels of the compact zones there are sarcomeres (S), leptomeres (L), and a few extromeres (E). At the level of the reticular zone there are reticulomeres (R = unisarcomeric muscle processes), leptomeres (L, not illustrated), and extromeres (E). The latter appear quantitatively with stretch. A summary of the measurements is graphically illustrated in Fig. 19.
tiation of the typical discharge patterns should therefore reside in part, at least, in the relationships between the nerve, the muscle, and their environments. The frog muscle spindle contains intrafusal muscle fibers each with a characteristic series of sensory zones (compact-reticular-compact). Katz (7) attributed dynamic and static responses to differences in elasticity and viscosity between reticular and compact zones. He assumed the neuromuscular contact to be relatively stable and the reticular zone to resist elongation beyond a limited degree of stretch. He suggested that the compact zones were largely responsible for the static response while the reticular zones induced mainly the dynamic response. He found it difficult to explain the offeffect at cessation of motor stimulation and the decline in response at maintained motor stimulation. Ottoson and Shepherd (9) observed with stroboscopic vital microscopy that the equatorial region of isolated frog spindles stretched about as much as the polar regions during the first 6-10 milliseconds of applied stretch. They concluded that the solution to the problem of stretch differentiation must be sought at the ultrastructural level. The basic aim for the present investigation was to describe the topographical changes that occur with passive stretch. For measurements of elongation changes, a reference structure is necessary. In the compact zone this "biological ruler" is the sarcomere. The entire reticular zone appears too variable in length to be considered as a measure of stretch and requires threedimensional analysis for measurements. However, a periodicity consisting of regular surface projections, previously reported to occur in this zone (4, 5) can be used. They are here referred to as reticulomeres. In addition, periodic leptomeric organelles (microladders) (3, 7) occur in both zones. These three "rulers" were used to quantitate passive
745
PASSIVE S T R E T C H IN F R O G SPINDLES PR
PS
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FIG. 2. Schematic representation of the experimental groups used in the stretch experiments. In'the S experiment (slackened) the toe joint was maximally flexed and the fascia cut (longitudinally) in order for the muscle to reach complete relaxation. In the PR experiment (physiological relaxation) the fascia was left intact and in the PS experiment (physiological stretch) the fascia was also intact and the toe joint maximally extended. The legs were pinned in these positions before incubation in,procaine and subsequent preservation in buffered glutaraldehyde.
stretch e l o n g a t i o n of b o t h zones within the p h y s i o l o g i c a l range using u l t r a s t r u c t u r a l technics. F i g u r e 1 illustrates these m e a s u r a b l e structures. I n the present investigation two p e r i o d i c structures have been given names. The reticulornere is a r a d i a l process f r o m the intrafusal fiber in the reticular zone c o n t a i n i n g only one sarcomere period. The extrornere is a hitherto u n r e p o r t e d p e r i o d i c structure which a p p e a r s in the extracellular space with stretch. The two m a i n prerequisites to q u a n t i t a t i o n were l o n g i t u d i n a l serial sectioning of the spindle a n d p r e v e n t i o n of fixative-induced muscle c o n t r a c t i o n .
MATERIAL AND METHODS The experiments were performed on the toe extensor muscle (Musculus extensor digitorum longus IV) of the frog (Rana pipiens). Muscle length measurements were attempted for the different groups (a) directly by visualization with a measuring magnifier and (b) indirectly by measurements of thin threads cut to the respective muscle lengths. The toe joint was pinned so that the muscle assumed slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) positions (Fig. 2). The tissue processing procedure was as follows: 1. Incubation in 1% procaine hydrochloride in frog Ringer solution for 15 minutes at about 5°C. 2. Fixation in 2.5 % glutaraldehyde in frog isotonic S6rensen's phosphate buffer overnight at about 5°C. 3. Osmication at room temperature for 5 minutes in 1% OsO4, buffered as under 2 (5).
746
KARLSSON, HOOKER AND BENDEICH
4. Isolation of spindles by microdissection in frog isotonic phosphate buffer ( > 10 spindles/ experimental group). 5. Postfixation for 60 minutes in 1% OsO~ buffered as under 2. 6. Acetone dehydration and Vestopal embedding. 7. Longitudinal serial sectioning of the entire sensory region with the knife edge parallel to the intrafusals. About 500 sections/spindle at 700-1100 A thermal feed on an LKB Ultrotome III were collected on Formvar films suspended on key hole grids. 8. Section staining with uranyl acetate and lead citrate. 9. Montage photography with a Siemens Elmiskop 101 at a calibrated magification of 2570 diameters. Some higher magnifications of details were also photographed. 10. Linear measurements and counts from the structures schematically drawn in Fig. 1 ( > 5 intrafusals/experimental group). 11. t-test for differences of means of all parameters in each experimental group. All measurements for any given parameter in each experimental group were pooled.
RESULTS W i t h stretch the muscles lengthen a n d become thinner. The muscle length changes were estimated to be in the ratio 1:1.1 : 1.3 for S, PR, a n d PS, respectively. Both qualitative a n d quantitative data were retrieved f r o m the material. I n general terms, the u l t r a s t r u c t u r a l quality of fixation was j u d g e d satisfactory for the aim of the investigation. The different zones are schematically illustrated in Fig. 3. U l t r a s t r u c t u r a l changes with stretch were observed in sarcomeres (Figs. 4-7), muscle nuclei (Figs. 8-10), reticulomeres (Figs. 11-13), extracellular material (Figs. 14-16), leptomeres (Figs. 17 a n d 18), internal capsule (Figs. 9 a n d 10), and sensory nerve endings (Figs. 9, 10, 12, and 13). F o r measurements, sarcomeres, reticulomeres, leptomeres, a n d extromeres were selected. M a x i m a l i n n e r capsule diameter was recorded for each specimen. Table I summarizes the m e a s u r e m e n t s made, their s t a n d a r d deviations a n d the t-test analyses
Fro. 3. Low magnification of a longitudinally sectioned sensory region of a muscle spindle from the PR experiment. Two intrafusals are seen. The lower intrafusal displays the three zones (compact = CZ, reticular = RZ, compact- CZ) from which measurements were obtained. A nuclear group is displayed in the reticular zone, which is about 50# long in this intrafusal unit. Myelinated fibers surround the two intrafusals on both sides of the reticular zones. In the upper part the outer capsule (OC) can be seen. Satellite cells (darker nuclei) appose the intrafusals in the left compact zones. Marker represents 10 #. About x 1 100. FIOS. 4-7. Sarcomeres from the S (Fig. 4), PR (Fig. 5), and PS (Figs. 6 and 7) experiments. Figs. 4-6 represent intrafusals and Fig. 7 an extrafusal sarcomere from the PS experiment [PS (EF)]. I-bands widen in the intrafusals with stretch [PS (IF)]. M-bands are prominent in all specimens. H zones are discernible in Figs. 6 and 7. There is no significant increase between S and PR groups. With stretch the sarcomere spacing increased 59 % from PR to PS (Fig. 19). No attempt was made to evaluate differences between intrafusals and extrafusals. About x 26 000. Micron marker in Fig. 6.
i
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748
KARLSSON~ H O O K E R A N D B E N D E I C H
TABLE I STATISTICAL SUMMARY OF MEASUREMENTS AND COMPARISONS Degree of Stretcha Reticulomere Statistic
Number Mean i n # Standard deviation t-Values for stretch comparisons PR- S PS PR
's
PR ps'
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"s
~'a
PS"
Leptomere
;
Va
inner Capsule Tube Width
Extromere
P;
~
PR
V;
"S PR PS"
85 166 64 147 668 214 2.4 2.7 4.! 1.6 1.5 2.4
111 324 157 0.148 0.14 0.16
23 46 247 0.084 0.081 0.081
0.6 1.0 1.1
0.002 0.02 0.04
0.009 0.007 0.007
2.7 2.0 2.1
- 2.6 b
- 1.3
0.65
2.4 b
0.3 0.1 0.2
- 0.89 9.7 o
100 b
13.8 b
0.16
7 11.7
15 8 11 8.1
-3.0 b
a S = slackened (with opened fascia); PR = physiological relaxation (with intact fascia); PS = Physiological stretch (with intact fascia). Means differ significantly at the 0.05 level or better. The t values were calculated from 4-digit numbers. The number of digits in the means reflect estimated precision of measurement.
f o r the differences of the m e a n s . F i g u r e 19 s h o w s g r a p h i c a l l y the p e r i o d i c i t y c h a n g e s a n d p e r c e n t a g e differences f o r the e x p e r i m e n t a l g r o u p s . T h e m u s c l e nuclei in t h e r e t i c u l a r z o n e w e r e closely a p p o s e d to e a c h o t h e r in the S a n d P R g r o u p s (Figs. 8 a n d 9). W i t h stretch (PS g r o u p ) t h e y a p p e a r e d to s e p a r a t e a n d f o r m a single c h a i n as illustrated in Fig. 10. S a r c o m e r e s in the c o m p a c t z o n e s c h a n g e t h e i r p e r i o d i c i t y w i t h s t r e t c h b u t o n l y f r o m P R to PS (Figs. 4-6). Little difference was o b s e r v e d b e t w e e n the i n t r a f u s a l s in the PS g r o u p a n d extrafusals of the s a m e g r o u p (Figs. 6 a n d 7). N o significant d i f f e r e n c e was f o u n d b e t w e e n S a n d P R g r o u p s in spite of a n e s t i m a t e d 10 % e l o n g a t i o n of the w h o l e m u s c l e (Fig. 19 a n d T a b l e I). R e t i c u l o m e r e s c o n s t i t u t e the m a i n p e r i o d i c s t r u c t u r e in the r e t i c u l a r z o n e (Figs. 8-10). T h e s e c o n t a i n e d o n e s a r c o m e r e w h i c h d i s p l a y e d n o I - b a n d in the S a n d P R FIos. 8-10. Examples of structural changes in the reticular zone from the S (Fig. 8), PR (Fig. 9), and PS (Fig. 10) experiments. Only a part of the reticular zone is illustrated. In Fig. 10 (lower part) a compact zone with a satellite cell exemplifies staggering of the reticular zones among the intrafusals in the same bundle. An obvious rearrangement of intrafusal nuclei (Nu) can be seen. They transform from a group to a string pattern with stretch. Note that the inner capsule (IC) displays irregularities in Fig. 9 (PR) that cannot be seen in Fig. 10 (PS). Also the decrease in inner capsule diameter is obvious with stretch. The intrafusals display regularly spaced processes (reticulomeres at arrows) that increase their spacing with stretch (compare Figs. 9 and 10). Sensory nerve endings often appose the top of the reticulomeres as illustrated at several locations in all figures. The nerve endings change shape (from rounded to elliptoid) with stretch (compare Figs. 9 and 10). About x 4 500.
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KARLSSON, HOOKERAND BENDEICH
groups (Figs. 11 and 12). With stretch (PS group), however, the I-band appeared and the outline of the process became "jagged" (Fig. 13). The reticulomere spacing increased significantly in each group (Fig. 19). The extracellular filamentous material within the internal capsule (Fig. 14) was characterized by r a n d o m orientation in the S and PR groups (Fig. 15). With maximal physiological stretch (PS), however, reorganization into a regular periodic pattern appeared in localized areas. This periodicity consisted of alternating dense and light lamellae associated with filamentous material (Fig. 16). After extensive search for these periodic structures (extromeres), a few were also found in the S and P R groups. However, a quantitative estimate revealed a more than 10-fold increase in occurrence with maximal physiological stretch (PS group). The periodicity (810 A) did not change significantly with stretch (Fig. 19). Leptomeres (Figs. 17 and 18) were observed in both compact and reticular zones in all specimens. It was estimated that about 100 leptomeres occurred in the sensory region of an intrafusal fiber. Their periodicity changed 13 % within the physiological range of passive stretch (Fig. 19). The inner capsule surrounded each intrafusal in the reticular zone and formed an open-ended sleeve in the compact zones. With stretch, this sleeve decreased in diameter (Fig. 19). Radially oriented irregularities in the lamellae were present in the P R group but seemed to have disappeared in the PS group (compare Figs. 9 and 14 with Fig. 10). The sensory nerve bulbs are longitudinally linked together along the sensory region. They changed from a relatively round profile in the S and PR groups to an elongated form in the PS group (compare Figs. 9, 10, 12, and 13).
DISCUSSION
Methodology Contraction of muscle is known to occur with immersion fixation in osmium tetroxide solutions. To prevent modification of the sarcomere length by g a m m a discharge or direct effects of the fixative, procaine incubation was used prior to fixaFXGS.11-13. Examples of reticulomeres from S (Fig. 11), PR (Fig. 12) and PS (Fig. 13) experiments. The reticular zone is characterized by intrafusal nuclei (Nu), reticulomeres (unisarcomeric muscle processes), and extraceUular filaments (E). In Fig. 11 two adjacent reticulomeres are seen. Their spacing increases 12 % (Fig. 19) between S (Fig. 11) and PR (Fig. 12) groups. Theadjacent reticulomere can be partially seen to the left in Fig. 12. Sensory nerve endings with rounded profiles (SE) appose the tops of the reticulomeres in Figs. 11 and 12. In Fig. 13 (PS) only one reticulomere is illustrated at this magnification. The spacing increases 53 % between PR and PS (Fig. 19). Its contours appear more "jagged", and I-filaments are distinct. The sensory nerve ending (SE) exhibits an elliptoid rather than rounded contour. Some extromeric patterns (under E) can be observed in the left part of Fig. 13 while the extracellular filaments appear randomly oriented in Figs. 11 and 12. About x 24 000.
752
KARLSSON, HOOKER AND BENDEICH
tion (8, 11). Glutaraldehyde fixation was employed since it has been shown that muscle retains its length after glutaraldehyde fixation regardless of imposed stretchrelease forces (2). This minimized artifacts caused by the dissection procedure. The muscle spindles were stretched in situ. We favored this a p p r o a c h since the extrafusal environment m a y be of importance with respect to the effect of passive stretch in the intrafusals. All measurements were pooled for each experimental group before the statistical analysis. Standard deviations were considerably larger for reticulomeres than any other measured periodicity. This m a y be explained by a biological variation between specimens and some obliquity differences between them at sectioning. Furthermore, the reticular zone periodicity is inherently variable. F o r example, the reticulomere interdistance decreases in the transition zone to allow merging with the series-coupled sarcomeres of the c o m p a c t zone. This is consistent with a slightly skewed distribution of measurements. The results of the statistical methods are therefore approximate rather than precise. The gross muscle lengths in the S, PR, and PS groups could only be approximated. This was due to difficulty in comparing measurements made in muscles with and without fascia. The experiment locations on the abscissa in Fig. 19 are therefore approximations. Oblique sectioning of the intrafusals increases the periodicity measurements. The S and P R groups would be more affected than the PS group by such errors since relaxed fibers m a y not be as straight as stretched ones. The means of the periodicities in the S and P R groups are generally lower than those in the PS group (Fig. 19). Sectioning errors would therefore only tend to decrease the significance level of differences between the groups. Compression at sectioning need not be considered since all periodicities were oriented perpendicular to the sectioning direction. Data Periodicities of certain extracellular and intracellular periodic structures were measured at slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) conditions of the muscle spindles. The changes of inner capsule diameter and the
FIGS. 14-16. Examples of the extracellular filaments in the reticular zone and their changes with stretch. Fig. 14 illustrates a tangential section through a reticular zone of a PR intrafusal unit. Note the density of extracellular filaments inside (ICS = inner capsular space) the inner capsule as compared to outside (OCS = outer capsular space). Figs. 15 and 16 illustrate the transformation of extracellular filaments from a random orientation in Fig. 15 (PR = physiological relaxation) to a periodic pattern in the inner capsule space (ICS) in Fig. 16 (PS = physiological stretch). In the lower parts of Figs. 15 and 16 the intrafusal (IF) is illustrated. Its contour changes with stretch. In the upper part of Fig. 16 a few sensory nerve bulbs (SE) are displayed. Fig. 14, about × 3 400; Figs. 15 and 16, about × 53 000. Marker in Fig. 14 = 10 #, in Figs. 15 and 16 ~ 1 f~.
754
KARLSSON, HOOKER AND BENDE[CH
Fins. 17 and 18. Examples of leptomeric spacings in PR (Fig. 17) and PS (Fig. 18) experiments. The leptomeres were sometimes observed near nuclei (Nu) as illustrated in Fig. 17, and they occurred in both reticular and compact zones. Little difference (13 %, see Fig. 19) was found in the spacings between these two experimental groups. As illustrated, the sarcomere lengths differed considerably About x 36 000. qualitative change of certain organelles were also considered. The percentage change with stretch (Fig. 19) was high for reticulomeres (reticular zone) and sarcomeres (compact zone), considerably less for leptomeres (both zones), and insignificant for extromeres (mainly outside the reticular zone). The inner capsule diameter decreased significantly with stretch. The conclusion for these data is that the percentage elongation of the reticular zone (reticulomeres) was at least equal to that of the compact zone (sarcomeres) during passive physiological stretch. Between S and PR, however, only the reticulomere spacing seemed to increase. This means that the reticular zone here m a y have offered less resistance to passive stretch than the compact zones. On the other hand, the indication of length change reversal between P R and PS (Fig. 19) may be due to a "tendon-like" property of the reticular zone in the PS group. This together with the
PASSIVE STRETCH IN F R O G SPINDLES
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FIG. 19. Diagram of measured changes in inner capsule diameter (IC WIDTH), RETICULOMERE spacings, SARCOMERE lengths, LEPTOMERE spacings and EXTROMERE spacings in slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) muscles according to Fig. 2. The ordinate is in microns. The abscissa denotes approximate length changes between experimental groups. Statistically insignificant changes at the 5 % level or better are dashed. Numbers outside parentheses refer to percentage changes occurring from the nearest group to the left. Numbers within parentheses refer to percentage changes occurring from S to PS groups. appearance of nonstretchable extromeres (Fig. 16) suggests that the reticular zone had reached near maximal elongation in the PS group. It is difficult to reconcile the lack of intrafusal sarcomere elongation with an estimated 10 % muscle elongation between S and P R (Fig. 19). Complementary elongation of sarcomeres in extracapsular spindle fibers and in nonsensory parts of the intrafusals m a y explain this difference. This suggests that the sensory region of the intrafusals responds differently to stretch than other parts of the muscle. This investigation has therefore presented evidence that the reticular zone is highly stretchable and remains stretched in the stationary phase. It is possible that it offers less resistance to stretch than the compact zone in the lower part of the physiological range. It may also reach maximal length within the physiological range. The fact that
756
KARLSSON, HOOKERAND BENDEICH
the reticular zone remains stretched during the stationary phase is difficult to reconcile with Katz's hypothesis (7) that it contributes relatively little to the static phase of the discharge pattern. The extromeres did not elongate with stretch (Fig. 19) but they increased in number. The nerve bulb chains are embedded in this material and the reticulomeres may be series-coupled by it. The transformation of extracellular filaments into extromeres could, therefore, reflect a protective mechanism against overstretch. This may be the mechanism Katz (7) anticipated. The presence of extromeres may reflect near-maximum length of the reticular and transition zones as well as the near-maximal length of the nerve bulb chains. Also, their appearance may reflect a stabilizing force that could affect the neuromuscular contacts. Such a mechanism could be basic for the understanding of transduction as well as the mechanistic differentiation of dynamic and static responses. It remains to determine the quantitative changes occurring in the nerve bulb chains and their topography at different degrees of stretch. This investigation is presently being pursued. Decrease in inner-capsule sleeve diameter, rearrangement of nuclei, and appearance of extromeres with stretch represent a combination of observations indicating that sizable forces are at play in and around the reticular zone. A vector of these forces may have an inward radial direction. The question then arises whether such forces participate in the generation of the electrical response. Obvious ultrastructural changes occur in the frog muscle spindle with physiological stimulation. It is conceivable that these changes could be utilized as instruments for a better understanding of the phenomenon of transduction. The authors wish to thank Dr John Edie for help with the statistical analysis and Miss Sharon Pratt for typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5.
6. 7.
8. 9. 10. 11.
EYZAGUIRRE,C., J. Neurophysiol. 21, 465 (1958). GALEY,F. R., J. Ultrastruet. Res. 26, 424 (1969). KARLSSON,U. and ANDERSSON-CED~RaREN,E., Y. Ultrastruet. Res. 23, 417 (1968) KARLSSON,U., A~DERSSON-CEDERGREN,E., and OTTOSON,D., Y. Ultrastruct. Res. 6, 136 (1962). -ibid. 14, 1 (1966). KATZ, B., Y. Physiol. (London) 111, 261 (1950). -Phil. Trans. Roy. Soe. London Ser. B 243, 221 (1961). MATTHEWS,P. B. C., and RUS~WORTH, G., J. Physiol. (London) 135, 263 (1957). OXTOSON,D. and SHEVHERD,G. M., Nature (London) 220, 912 (1968). SnEVnERD,G. M. and OTTOSOZ~,D., CoM Spring Harbor Symp. Quant. Biol. 30, 95 (1965), SJ6STRAND,F. S., Nature (London) 201, 45 (1964).
36, 743-756 (1971)
743
Quantitative Changes in the Frog Muscle Spindle with Passive Stretch ~ ULF L. KARLSSON,2 WILLIAM M. HOOKER,3 and ELIZABETH G. BENDEICH2
Dental Research Laboratory, College of Dentistry, and Department of Anatomy, College of Medicine, University of Iowa, Iowa City 52240 Received December 30, 1970 Ultrastructural changes in the frog intrafusal were qualitatively and quantitatively evaluated with passive stretch. The following observations were made: (1) The reticular zone was stretchable and remained stretched in the stationary phase. (2) Reticulomeres (unisarcomeric muscle processes in the reticular zone) appeared to be series-coupled with the extracellular material. (3) Extracellular filamentous material transformed from a random orientation to a nonstretchable periodic (810 A) pattern (extromeres). (4) The inner capsule sleeve decreased in diameter. (5) Groups of intrafusal nuclei rearranged from a group to a string pattern. (6) Nerve bulbs changed their contours from rounded to elliptoid. (7) Leptomere spacings increased less than sarcomere spacings. The significance of these changes is discussed in terms of spindle function. The muscle spindle responds to stretch with a characteristic discharge pattern that is divided into a dynamic and a static phase (6). The dynamic peak frequency increases with greater initial lengths of the spindle as well as with increased stretch velocity (10). The static phase frequency increases with increasing lengths of the spindle. Other characteristics are an off-effect at cessation of m o t o r stimulation and a decline of the response at maintained m o t o r stimulation (1). The basic mechanism for these discharge patterns is unknown. Mechanical stretch is the stimulus ultimately governing the net electrical response. U n d o u b t e d l y the morphological organization determines this response. The differen1 This investigation was supported by the General Research Support Grant to University of Iowa, Colleges of Dentistry and Medicine and the Neurosensory Center, Program Project Grant Number NSO3354 of the National Institute of Neurological Diseases and Stroke. Neurosensory Center, Publication 221. 2 Present address: Dental Research Laboratory, College of Dentistry, University of Iowa, Iowa City, Iowa 52240. 3 Present address: Department of Anatomy, School of Medicine, Loma Linda University, Loma Linda, California 92354.
744
KARLSSON, HOOKER AND BENDEICH C o m p a c t zone
;~=
: 27( - "
R e t i c u l a r zone
.~-'. 2 2 " 5 2 ; : : - 7~ E ~ "" E x t r a c e l l u l a r ~ - .J .:'-'~.~'-'2.; - 2 . "-V-:2£'} : r
','''c"
I' s--q
~
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,oo,.~.
FIo. 1. Schematic representation of measurable periodicities in and around the compact and reticular zones of an intrafusal unit in the frog muscle spindle. At the levels of the compact zones there are sarcomeres (S), leptomeres (L), and a few extromeres (E). At the level of the reticular zone there are reticulomeres (R = unisarcomeric muscle processes), leptomeres (L, not illustrated), and extromeres (E). The latter appear quantitatively with stretch. A summary of the measurements is graphically illustrated in Fig. 19.
tiation of the typical discharge patterns should therefore reside in part, at least, in the relationships between the nerve, the muscle, and their environments. The frog muscle spindle contains intrafusal muscle fibers each with a characteristic series of sensory zones (compact-reticular-compact). Katz (7) attributed dynamic and static responses to differences in elasticity and viscosity between reticular and compact zones. He assumed the neuromuscular contact to be relatively stable and the reticular zone to resist elongation beyond a limited degree of stretch. He suggested that the compact zones were largely responsible for the static response while the reticular zones induced mainly the dynamic response. He found it difficult to explain the offeffect at cessation of motor stimulation and the decline in response at maintained motor stimulation. Ottoson and Shepherd (9) observed with stroboscopic vital microscopy that the equatorial region of isolated frog spindles stretched about as much as the polar regions during the first 6-10 milliseconds of applied stretch. They concluded that the solution to the problem of stretch differentiation must be sought at the ultrastructural level. The basic aim for the present investigation was to describe the topographical changes that occur with passive stretch. For measurements of elongation changes, a reference structure is necessary. In the compact zone this "biological ruler" is the sarcomere. The entire reticular zone appears too variable in length to be considered as a measure of stretch and requires threedimensional analysis for measurements. However, a periodicity consisting of regular surface projections, previously reported to occur in this zone (4, 5) can be used. They are here referred to as reticulomeres. In addition, periodic leptomeric organelles (microladders) (3, 7) occur in both zones. These three "rulers" were used to quantitate passive
745
PASSIVE S T R E T C H IN F R O G SPINDLES PR
PS
2 seia Bone-
FIG. 2. Schematic representation of the experimental groups used in the stretch experiments. In'the S experiment (slackened) the toe joint was maximally flexed and the fascia cut (longitudinally) in order for the muscle to reach complete relaxation. In the PR experiment (physiological relaxation) the fascia was left intact and in the PS experiment (physiological stretch) the fascia was also intact and the toe joint maximally extended. The legs were pinned in these positions before incubation in,procaine and subsequent preservation in buffered glutaraldehyde.
stretch e l o n g a t i o n of b o t h zones within the p h y s i o l o g i c a l range using u l t r a s t r u c t u r a l technics. F i g u r e 1 illustrates these m e a s u r a b l e structures. I n the present investigation two p e r i o d i c structures have been given names. The reticulornere is a r a d i a l process f r o m the intrafusal fiber in the reticular zone c o n t a i n i n g only one sarcomere period. The extrornere is a hitherto u n r e p o r t e d p e r i o d i c structure which a p p e a r s in the extracellular space with stretch. The two m a i n prerequisites to q u a n t i t a t i o n were l o n g i t u d i n a l serial sectioning of the spindle a n d p r e v e n t i o n of fixative-induced muscle c o n t r a c t i o n .
MATERIAL AND METHODS The experiments were performed on the toe extensor muscle (Musculus extensor digitorum longus IV) of the frog (Rana pipiens). Muscle length measurements were attempted for the different groups (a) directly by visualization with a measuring magnifier and (b) indirectly by measurements of thin threads cut to the respective muscle lengths. The toe joint was pinned so that the muscle assumed slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) positions (Fig. 2). The tissue processing procedure was as follows: 1. Incubation in 1% procaine hydrochloride in frog Ringer solution for 15 minutes at about 5°C. 2. Fixation in 2.5 % glutaraldehyde in frog isotonic S6rensen's phosphate buffer overnight at about 5°C. 3. Osmication at room temperature for 5 minutes in 1% OsO4, buffered as under 2 (5).
746
KARLSSON, HOOKER AND BENDEICH
4. Isolation of spindles by microdissection in frog isotonic phosphate buffer ( > 10 spindles/ experimental group). 5. Postfixation for 60 minutes in 1% OsO~ buffered as under 2. 6. Acetone dehydration and Vestopal embedding. 7. Longitudinal serial sectioning of the entire sensory region with the knife edge parallel to the intrafusals. About 500 sections/spindle at 700-1100 A thermal feed on an LKB Ultrotome III were collected on Formvar films suspended on key hole grids. 8. Section staining with uranyl acetate and lead citrate. 9. Montage photography with a Siemens Elmiskop 101 at a calibrated magification of 2570 diameters. Some higher magnifications of details were also photographed. 10. Linear measurements and counts from the structures schematically drawn in Fig. 1 ( > 5 intrafusals/experimental group). 11. t-test for differences of means of all parameters in each experimental group. All measurements for any given parameter in each experimental group were pooled.
RESULTS W i t h stretch the muscles lengthen a n d become thinner. The muscle length changes were estimated to be in the ratio 1:1.1 : 1.3 for S, PR, a n d PS, respectively. Both qualitative a n d quantitative data were retrieved f r o m the material. I n general terms, the u l t r a s t r u c t u r a l quality of fixation was j u d g e d satisfactory for the aim of the investigation. The different zones are schematically illustrated in Fig. 3. U l t r a s t r u c t u r a l changes with stretch were observed in sarcomeres (Figs. 4-7), muscle nuclei (Figs. 8-10), reticulomeres (Figs. 11-13), extracellular material (Figs. 14-16), leptomeres (Figs. 17 a n d 18), internal capsule (Figs. 9 a n d 10), and sensory nerve endings (Figs. 9, 10, 12, and 13). F o r measurements, sarcomeres, reticulomeres, leptomeres, a n d extromeres were selected. M a x i m a l i n n e r capsule diameter was recorded for each specimen. Table I summarizes the m e a s u r e m e n t s made, their s t a n d a r d deviations a n d the t-test analyses
Fro. 3. Low magnification of a longitudinally sectioned sensory region of a muscle spindle from the PR experiment. Two intrafusals are seen. The lower intrafusal displays the three zones (compact = CZ, reticular = RZ, compact- CZ) from which measurements were obtained. A nuclear group is displayed in the reticular zone, which is about 50# long in this intrafusal unit. Myelinated fibers surround the two intrafusals on both sides of the reticular zones. In the upper part the outer capsule (OC) can be seen. Satellite cells (darker nuclei) appose the intrafusals in the left compact zones. Marker represents 10 #. About x 1 100. FIOS. 4-7. Sarcomeres from the S (Fig. 4), PR (Fig. 5), and PS (Figs. 6 and 7) experiments. Figs. 4-6 represent intrafusals and Fig. 7 an extrafusal sarcomere from the PS experiment [PS (EF)]. I-bands widen in the intrafusals with stretch [PS (IF)]. M-bands are prominent in all specimens. H zones are discernible in Figs. 6 and 7. There is no significant increase between S and PR groups. With stretch the sarcomere spacing increased 59 % from PR to PS (Fig. 19). No attempt was made to evaluate differences between intrafusals and extrafusals. About x 26 000. Micron marker in Fig. 6.
i
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748
KARLSSON~ H O O K E R A N D B E N D E I C H
TABLE I STATISTICAL SUMMARY OF MEASUREMENTS AND COMPARISONS Degree of Stretcha Reticulomere Statistic
Number Mean i n # Standard deviation t-Values for stretch comparisons PR- S PS PR
's
PR ps'
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"s
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;
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inner Capsule Tube Width
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P;
~
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V;
"S PR PS"
85 166 64 147 668 214 2.4 2.7 4.! 1.6 1.5 2.4
111 324 157 0.148 0.14 0.16
23 46 247 0.084 0.081 0.081
0.6 1.0 1.1
0.002 0.02 0.04
0.009 0.007 0.007
2.7 2.0 2.1
- 2.6 b
- 1.3
0.65
2.4 b
0.3 0.1 0.2
- 0.89 9.7 o
100 b
13.8 b
0.16
7 11.7
15 8 11 8.1
-3.0 b
a S = slackened (with opened fascia); PR = physiological relaxation (with intact fascia); PS = Physiological stretch (with intact fascia). Means differ significantly at the 0.05 level or better. The t values were calculated from 4-digit numbers. The number of digits in the means reflect estimated precision of measurement.
f o r the differences of the m e a n s . F i g u r e 19 s h o w s g r a p h i c a l l y the p e r i o d i c i t y c h a n g e s a n d p e r c e n t a g e differences f o r the e x p e r i m e n t a l g r o u p s . T h e m u s c l e nuclei in t h e r e t i c u l a r z o n e w e r e closely a p p o s e d to e a c h o t h e r in the S a n d P R g r o u p s (Figs. 8 a n d 9). W i t h stretch (PS g r o u p ) t h e y a p p e a r e d to s e p a r a t e a n d f o r m a single c h a i n as illustrated in Fig. 10. S a r c o m e r e s in the c o m p a c t z o n e s c h a n g e t h e i r p e r i o d i c i t y w i t h s t r e t c h b u t o n l y f r o m P R to PS (Figs. 4-6). Little difference was o b s e r v e d b e t w e e n the i n t r a f u s a l s in the PS g r o u p a n d extrafusals of the s a m e g r o u p (Figs. 6 a n d 7). N o significant d i f f e r e n c e was f o u n d b e t w e e n S a n d P R g r o u p s in spite of a n e s t i m a t e d 10 % e l o n g a t i o n of the w h o l e m u s c l e (Fig. 19 a n d T a b l e I). R e t i c u l o m e r e s c o n s t i t u t e the m a i n p e r i o d i c s t r u c t u r e in the r e t i c u l a r z o n e (Figs. 8-10). T h e s e c o n t a i n e d o n e s a r c o m e r e w h i c h d i s p l a y e d n o I - b a n d in the S a n d P R FIos. 8-10. Examples of structural changes in the reticular zone from the S (Fig. 8), PR (Fig. 9), and PS (Fig. 10) experiments. Only a part of the reticular zone is illustrated. In Fig. 10 (lower part) a compact zone with a satellite cell exemplifies staggering of the reticular zones among the intrafusals in the same bundle. An obvious rearrangement of intrafusal nuclei (Nu) can be seen. They transform from a group to a string pattern with stretch. Note that the inner capsule (IC) displays irregularities in Fig. 9 (PR) that cannot be seen in Fig. 10 (PS). Also the decrease in inner capsule diameter is obvious with stretch. The intrafusals display regularly spaced processes (reticulomeres at arrows) that increase their spacing with stretch (compare Figs. 9 and 10). Sensory nerve endings often appose the top of the reticulomeres as illustrated at several locations in all figures. The nerve endings change shape (from rounded to elliptoid) with stretch (compare Figs. 9 and 10). About x 4 500.
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groups (Figs. 11 and 12). With stretch (PS group), however, the I-band appeared and the outline of the process became "jagged" (Fig. 13). The reticulomere spacing increased significantly in each group (Fig. 19). The extracellular filamentous material within the internal capsule (Fig. 14) was characterized by r a n d o m orientation in the S and PR groups (Fig. 15). With maximal physiological stretch (PS), however, reorganization into a regular periodic pattern appeared in localized areas. This periodicity consisted of alternating dense and light lamellae associated with filamentous material (Fig. 16). After extensive search for these periodic structures (extromeres), a few were also found in the S and P R groups. However, a quantitative estimate revealed a more than 10-fold increase in occurrence with maximal physiological stretch (PS group). The periodicity (810 A) did not change significantly with stretch (Fig. 19). Leptomeres (Figs. 17 and 18) were observed in both compact and reticular zones in all specimens. It was estimated that about 100 leptomeres occurred in the sensory region of an intrafusal fiber. Their periodicity changed 13 % within the physiological range of passive stretch (Fig. 19). The inner capsule surrounded each intrafusal in the reticular zone and formed an open-ended sleeve in the compact zones. With stretch, this sleeve decreased in diameter (Fig. 19). Radially oriented irregularities in the lamellae were present in the P R group but seemed to have disappeared in the PS group (compare Figs. 9 and 14 with Fig. 10). The sensory nerve bulbs are longitudinally linked together along the sensory region. They changed from a relatively round profile in the S and PR groups to an elongated form in the PS group (compare Figs. 9, 10, 12, and 13).
DISCUSSION
Methodology Contraction of muscle is known to occur with immersion fixation in osmium tetroxide solutions. To prevent modification of the sarcomere length by g a m m a discharge or direct effects of the fixative, procaine incubation was used prior to fixaFXGS.11-13. Examples of reticulomeres from S (Fig. 11), PR (Fig. 12) and PS (Fig. 13) experiments. The reticular zone is characterized by intrafusal nuclei (Nu), reticulomeres (unisarcomeric muscle processes), and extraceUular filaments (E). In Fig. 11 two adjacent reticulomeres are seen. Their spacing increases 12 % (Fig. 19) between S (Fig. 11) and PR (Fig. 12) groups. Theadjacent reticulomere can be partially seen to the left in Fig. 12. Sensory nerve endings with rounded profiles (SE) appose the tops of the reticulomeres in Figs. 11 and 12. In Fig. 13 (PS) only one reticulomere is illustrated at this magnification. The spacing increases 53 % between PR and PS (Fig. 19). Its contours appear more "jagged", and I-filaments are distinct. The sensory nerve ending (SE) exhibits an elliptoid rather than rounded contour. Some extromeric patterns (under E) can be observed in the left part of Fig. 13 while the extracellular filaments appear randomly oriented in Figs. 11 and 12. About x 24 000.
752
KARLSSON, HOOKER AND BENDEICH
tion (8, 11). Glutaraldehyde fixation was employed since it has been shown that muscle retains its length after glutaraldehyde fixation regardless of imposed stretchrelease forces (2). This minimized artifacts caused by the dissection procedure. The muscle spindles were stretched in situ. We favored this a p p r o a c h since the extrafusal environment m a y be of importance with respect to the effect of passive stretch in the intrafusals. All measurements were pooled for each experimental group before the statistical analysis. Standard deviations were considerably larger for reticulomeres than any other measured periodicity. This m a y be explained by a biological variation between specimens and some obliquity differences between them at sectioning. Furthermore, the reticular zone periodicity is inherently variable. F o r example, the reticulomere interdistance decreases in the transition zone to allow merging with the series-coupled sarcomeres of the c o m p a c t zone. This is consistent with a slightly skewed distribution of measurements. The results of the statistical methods are therefore approximate rather than precise. The gross muscle lengths in the S, PR, and PS groups could only be approximated. This was due to difficulty in comparing measurements made in muscles with and without fascia. The experiment locations on the abscissa in Fig. 19 are therefore approximations. Oblique sectioning of the intrafusals increases the periodicity measurements. The S and P R groups would be more affected than the PS group by such errors since relaxed fibers m a y not be as straight as stretched ones. The means of the periodicities in the S and P R groups are generally lower than those in the PS group (Fig. 19). Sectioning errors would therefore only tend to decrease the significance level of differences between the groups. Compression at sectioning need not be considered since all periodicities were oriented perpendicular to the sectioning direction. Data Periodicities of certain extracellular and intracellular periodic structures were measured at slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) conditions of the muscle spindles. The changes of inner capsule diameter and the
FIGS. 14-16. Examples of the extracellular filaments in the reticular zone and their changes with stretch. Fig. 14 illustrates a tangential section through a reticular zone of a PR intrafusal unit. Note the density of extracellular filaments inside (ICS = inner capsular space) the inner capsule as compared to outside (OCS = outer capsular space). Figs. 15 and 16 illustrate the transformation of extracellular filaments from a random orientation in Fig. 15 (PR = physiological relaxation) to a periodic pattern in the inner capsule space (ICS) in Fig. 16 (PS = physiological stretch). In the lower parts of Figs. 15 and 16 the intrafusal (IF) is illustrated. Its contour changes with stretch. In the upper part of Fig. 16 a few sensory nerve bulbs (SE) are displayed. Fig. 14, about × 3 400; Figs. 15 and 16, about × 53 000. Marker in Fig. 14 = 10 #, in Figs. 15 and 16 ~ 1 f~.
754
KARLSSON, HOOKER AND BENDE[CH
Fins. 17 and 18. Examples of leptomeric spacings in PR (Fig. 17) and PS (Fig. 18) experiments. The leptomeres were sometimes observed near nuclei (Nu) as illustrated in Fig. 17, and they occurred in both reticular and compact zones. Little difference (13 %, see Fig. 19) was found in the spacings between these two experimental groups. As illustrated, the sarcomere lengths differed considerably About x 36 000. qualitative change of certain organelles were also considered. The percentage change with stretch (Fig. 19) was high for reticulomeres (reticular zone) and sarcomeres (compact zone), considerably less for leptomeres (both zones), and insignificant for extromeres (mainly outside the reticular zone). The inner capsule diameter decreased significantly with stretch. The conclusion for these data is that the percentage elongation of the reticular zone (reticulomeres) was at least equal to that of the compact zone (sarcomeres) during passive physiological stretch. Between S and PR, however, only the reticulomere spacing seemed to increase. This means that the reticular zone here m a y have offered less resistance to passive stretch than the compact zones. On the other hand, the indication of length change reversal between P R and PS (Fig. 19) may be due to a "tendon-like" property of the reticular zone in the PS group. This together with the
PASSIVE STRETCH IN F R O G SPINDLES
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FIG. 19. Diagram of measured changes in inner capsule diameter (IC WIDTH), RETICULOMERE spacings, SARCOMERE lengths, LEPTOMERE spacings and EXTROMERE spacings in slackened (S), physiologically relaxed (PR), and physiologically stretched (PS) muscles according to Fig. 2. The ordinate is in microns. The abscissa denotes approximate length changes between experimental groups. Statistically insignificant changes at the 5 % level or better are dashed. Numbers outside parentheses refer to percentage changes occurring from the nearest group to the left. Numbers within parentheses refer to percentage changes occurring from S to PS groups. appearance of nonstretchable extromeres (Fig. 16) suggests that the reticular zone had reached near maximal elongation in the PS group. It is difficult to reconcile the lack of intrafusal sarcomere elongation with an estimated 10 % muscle elongation between S and P R (Fig. 19). Complementary elongation of sarcomeres in extracapsular spindle fibers and in nonsensory parts of the intrafusals m a y explain this difference. This suggests that the sensory region of the intrafusals responds differently to stretch than other parts of the muscle. This investigation has therefore presented evidence that the reticular zone is highly stretchable and remains stretched in the stationary phase. It is possible that it offers less resistance to stretch than the compact zone in the lower part of the physiological range. It may also reach maximal length within the physiological range. The fact that
756
KARLSSON, HOOKERAND BENDEICH
the reticular zone remains stretched during the stationary phase is difficult to reconcile with Katz's hypothesis (7) that it contributes relatively little to the static phase of the discharge pattern. The extromeres did not elongate with stretch (Fig. 19) but they increased in number. The nerve bulb chains are embedded in this material and the reticulomeres may be series-coupled by it. The transformation of extracellular filaments into extromeres could, therefore, reflect a protective mechanism against overstretch. This may be the mechanism Katz (7) anticipated. The presence of extromeres may reflect near-maximum length of the reticular and transition zones as well as the near-maximal length of the nerve bulb chains. Also, their appearance may reflect a stabilizing force that could affect the neuromuscular contacts. Such a mechanism could be basic for the understanding of transduction as well as the mechanistic differentiation of dynamic and static responses. It remains to determine the quantitative changes occurring in the nerve bulb chains and their topography at different degrees of stretch. This investigation is presently being pursued. Decrease in inner-capsule sleeve diameter, rearrangement of nuclei, and appearance of extromeres with stretch represent a combination of observations indicating that sizable forces are at play in and around the reticular zone. A vector of these forces may have an inward radial direction. The question then arises whether such forces participate in the generation of the electrical response. Obvious ultrastructural changes occur in the frog muscle spindle with physiological stimulation. It is conceivable that these changes could be utilized as instruments for a better understanding of the phenomenon of transduction. The authors wish to thank Dr John Edie for help with the statistical analysis and Miss Sharon Pratt for typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5.
6. 7.
8. 9. 10. 11.
EYZAGUIRRE,C., J. Neurophysiol. 21, 465 (1958). GALEY,F. R., J. Ultrastruet. Res. 26, 424 (1969). KARLSSON,U. and ANDERSSON-CED~RaREN,E., Y. Ultrastruet. Res. 23, 417 (1968) KARLSSON,U., A~DERSSON-CEDERGREN,E., and OTTOSON,D., Y. Ultrastruct. Res. 6, 136 (1962). -ibid. 14, 1 (1966). KATZ, B., Y. Physiol. (London) 111, 261 (1950). -Phil. Trans. Roy. Soe. London Ser. B 243, 221 (1961). MATTHEWS,P. B. C., and RUS~WORTH, G., J. Physiol. (London) 135, 263 (1957). OXTOSON,D. and SHEVHERD,G. M., Nature (London) 220, 912 (1968). SnEVnERD,G. M. and OTTOSOZ~,D., CoM Spring Harbor Symp. Quant. Biol. 30, 95 (1965), SJ6STRAND,F. S., Nature (London) 201, 45 (1964).