Development and maintenance of dendrite bundles after cordotomy in exercised and nonexercised cats

EXPERIMENTAL

NEUROLOGY

76,428-440

(1982)

Development and Maintenance of Dendrite Bundles after Cordotomy in Exercised and Nonexercised Cats PATRICIA A. REBACK, ARNOLD B. SCHEIBEL, AND JUDITH L. SMITH' Departments

of Kinesiology and Anatomy, and the Brain Research University of California, Los Angeles, California 90024 Received

December

I!,

Institute,

I981

Alpha motoneuron dendrite bundle formations were examined in the lumbar cord of kittens which had experienced Tr2 transection at either 2 or 12 weeks of age. Animals in both age groups were maintained on the average of 4 months postcordotomy and further subdivided as follows: one group experienced daily treadmill exercise for 20 to 30 min; the other group was unexercised. The effect of cordotomy on degree and complexity of bundling in all four groups was observed. Bundle complexity was also compared with ability to walk on a motorized treadmill. The dendrite bundles at the lumbar level of all kittens spinalized at 2 weeks were immature while bundles at the cervical level had matured normally. The dendrite bundles at the lumbar level for the cats spinalized at 12 weeks showed signs of degeneration. Neither exercise training nor walking ability was correlated with the complexity of dendrite bundling at the lumbar level. These results indicate that an intact cord is necessary for the maturation of bundles in the kittens spinalized at 2 weeks and for maintenance of existing bundle formations.

INTRODUCTION Alpha motoneuron dendrite bundles have been observed in the cervical and lumbosacral spinal cord of many mammals ( 1, 16, 29). These formations extend in a rostral-caudal direction and can project for several millimeters in length. Characteristically, dendrites of a given motoneuron may enter into different bundles and any one bundle may include dendrites from motoneurons of different functional groups. Dendrite bundles make Abbreviations: Eaxercised, NE-nonexercised. ’ This work was supported by grants NS 16333 and NS 10423 from the National Institutes of Health. Please send reprint requests to Dr. Reback, Department of Kinesiology, 2875 Slichter Hall, UCLA, Los Angeles, CA 90024. 428 0014-4886/82/050428-13%02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any fom reserved.

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their appearance in the lumbosacral cord on postnatal days 12 through 14, and coincident with the onset of reciprocal activity in flexor-extensor muscle pairs in the hind limbs, their maturation parallels development of greater weight-bearing and walking activities (28, 29). By the fourth postnatal month, both bundles and walking patterns resemble those of the adult cat. The coeval development of dendrite bundles and walking ability suggested (28, 29) that the alpha motoneuron dendrite bundles may provide one of the neuronal substrates necessary for developing appropriate reciprocal activity between various muscle groups involved in hind-limb activity. To test this hypothesis, we selected the cordotomized kitten, which has shown extreme variability in quadrupedal movements (8). Some kittens subjected to low thoracic spinal transection later were able to support themselves on all four limbs, produce rhythmical flexion and extension movements, and more complex postural adjustments (30,33). Others, however, merely propel themselves ahead with the forelimbs while dragging the hind-limbs along (30, 33). This variability can also be seen in treadmill walking where the hind-limbs are placed on the treadmill while the forelimbs are positioned on a platform (3 3). As mentioned in a preliminary report (25), we assessed the degree of dendrite bundle development in the kitten with a cordotomy compared with the normal kitten and looked for correlation between treadmill walking ability and degree of bundle development. Because transections in increasingly mature animals are reported to have more negative effects on the degree of recovery of walking ability (4, 30) a comparison was made between cats with cordotomies at 2 and 12 weeks of age. Also, cutaneous stimulation (30) and regular training [ (2 1 ), and see Methods below] were reported, respectively, to aid in the establishment of hind-limb walking and to decrease the amount of neuronal atrophy in spinalized animals. Thus it was decided to look more closely at the effect of exercise on development and maintenance of the dendrite bundles in the cordotomized animals of both age groups. METHODS Spinal cord transections at T12 were made during two stages of development: (i) 2 weeks postnatal, when the spinal cord is morphologically immature and flexor-extensor movements are rudimentary, and (ii) 12 weeks postnatal, when dendrite bundles and walking ability approach that of the mature cat (28, 29). Each age group was separated into two subgroups: those exercised daily for 20 to 30 min of walking on a motorized treadmill (2-E and 12-E) and those not exercised (2-NE, 12-NE), but allowed to move spontaneously in a cage; see Smith et al, (33) for details.

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The cats were maintained for an average of 4 months (11 to 19 weeks) postcordotomy. Prior to killing, both nonexercised and exercised groups were rated on spontaneous activity and ability to walk on a motorized treadmill at several weeks. A rating scale is described in the previous paper (33). The cats were killed under anesthesia by perfusion fixation. The thorax was opened and a cut was made in the right atrium. The left ventricle was punctured and a needle inserted into the ascending aorta. The animals were first perfused with phosphate-buffered saline (at 37°C). After the saline rinse, the animals were perfused with 500 to 900 ml phosphatebuffered 10% Formalin at pH 7.2. The cord was removed immediately and cut transversely into l-cm blocks. After removal of the dura, the cord specimens were placed 24 h in buffered Formalin at 4°C. The leptomeninges were removed and the tissue was processed using the rapid Golgi technique (28).

FIG. 1. Schematic drawings representing cord sections. See text for a description

the degree of bundling of the rating scale.

observed

in the lumbrosactal

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Horizontal cuts of 120 pm were made from six or seven divisions of the lumbosacral cord, each 1 cm in length yielding approximately 150 to 175 sections per animal. For control purposes similarly oriented blocks of tissue were prepared from the cervical region of the cord, usually C2 through CS. All slides were encoded with information which included a numbered reference to each animal, the anatomic locus of the block, and the serial order within the block. These precautions were taken to prevent observer bias. Each animal was rated on a 1 to 5 scale for complexity of bundles. Establishment of an overall grade for each animal was determined by averaging the ratings for each section of material. If no bundles were seen in any section, a numerical rating of 0 was given to the animal. A numerical rating of 4 or 5 is equivalent to bundle morphology of the normal mature 3-month-old cat as described by Scheibel and Scheibel (28). The rating criteria are described below and examples are illustrated in Fig. 1. O-No bundles in any section. l-Minimal evidence of dendrite bundles with at least one alpha-motoneuron soma (Fig. la). 2-At least one well formed bundle with three dendrites and the alpha motoneuron present; no length requirement (see Fig. lb). ~-TWO to three bundles per section with the alpha motoneuron present; more than three dendrites per bundle projecting at least 0.5 mm. Here the length is more important than the number of dendrites per bundle due to variation in stain intensity (Fig. 1c). 4-Three bundles with at least five dendrites per bundle extending 0.5 to 1 mm (Fig. Id). 5-Similar to rating 4 but with dendrites of more than 1 mm in length (not illustrated). These ratings were then compared with ratings of locomotor performance on the motorized treadmill. The significance of daily exercise on development or maintenance of the dendrite bundles was also analyzed statistically using the Mann-Whitney U test. RESULTS Evaluation of the various rating criteria for all animals is summarized in Table 1. Treadmill ratings varied widely for both age groups, as did the complexity of dendrite bundle formation. The degree of bundle complexity, however, did not appear to be related to the quality of treadmill locomotion. Complexity of bundles ranged from a rating of 0 (no bundles) to 3 (two to three bundles per section). Although a few individual sections showed complexity equivalent to that seen in the 3-month-old cat (ratings 4 and 5), no overall rating was greater than 3 for any animal. Exercise appeared to have no direct effect on development or maintenance of the bundles in either age group. Bundle Development and Maintenance. Integrity of the central nervous

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Ratings for Spinalixed Kittens at Time of Killing Treatment and cat no. 2-NE 30 130 132 170 2-E 32 110 131 12-NE 135 240 222 12-E 71 72 80 173 175

Dendrite bundles

Treadmill

0 1 2 3

NR 6.6 NR 4.8

0 2 1

NR 3.0 5.2

3 2 1

2.0 1.0 1.9

3 2 3 2 2

1.4 7.6 4.5 8.0 2.0

ratit&

“See Smith ef al. (33) for rating scale; a rating of 9 is the highest denoting consistent walking with good weight support. NR, not rated because of joint subluxations.

system is not a prerequisite for rudimentary dendrite bundle formation in the spinal cord, but does appear to be important for bundle maturation. Two of the animals examined from the 2-week cordotomized groups had virtually no bundling in the lumbosacral region (Fig. 2a). The remaining lumbosacral cords of the animals cordotomized at 2 weeks of age did contain many sections with dendrite bundles. Most bundles, however, were 0.5 mm or less in length and contained at most three to five dendrites; no sections showed ratings higher than 3. Figure 2b is a representative sample of these sections. It seems unlikely that this material was inadequately impregnated as the control cervical levels, which were processed along with the lumbosacral blocks, showed good impregnation and very complete bundling (Fig. 2~). All animals cordotomized at 2 weeks were killed at an age at which dendrite bundles are fully developed (ratings 4 and 5) in the normal kitten (28). In animals cordotomized at 12 weeks of age, subsequent development appeared arrested and there was evidence of progressive deterioration of dendrite bundles. The normal 1Zweek postnatal cat shows quite complex

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FIG. 2. Representative photomicrographs of Golgi stains showing degrees of bundling. aA lumbar section (L6) from a cat cordotomized at 2 weeks of age (2-E) that had no observable bundling. b-A more typical lumbar section (L6) from a 2-E cat showed moderate bundling that was rated a 2. c-A cervical (control) section (C,) of the cord from the same animal as in b demonstrated complex, mature bundling typical of the highest rating of 5. d-A lumbar section (L6) from a 2-E animal showing intermediate bundling that was rated 3.

bundling with many bundles per section, coursing more than 1 mm in length (29). In comparison, 12-week cordotomized cats showed bundles of diminished length and complexity. Ratings for these animals ranged from

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no bundles at all (0) to bundles of short length (0.5 mm or less) containing three to five dendrites (rating 3) (Fig. 2d). There were also visible signs of degeneration in the dendrites themselves such as nodular deformities and fragmented segments of dendrite shafts. These animals were all killed at an age of 7 months (or older) when bundle complexity should have been equivalent to the adult cat (rating 5). The low ratings, however, of all cats in this grouping (0 through 3) indicates

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2 (Continued)

that this maturation did not occur and that there might have been degeneration of bundles from their initial states as well. TreadmiiI Exercise. Daily treadmill exercise had no positive effect on bundle formation or maintenance. In the 2-week cordotomized cats (both 2-E and 2-NE) one animal in each group showed no bundle formation. Ratings were actually higher for two of four animals in the nonexercised group compared with the animals in the exercised group. Statistical analysis by the Mann-Whitney U test, however, showed no significant difference

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FIGURE 2 (ConGnd)

between these groups at the (Y= 0.05 level. Similarly, on the data collec :ted for the 12-week cordotomized group, there was no statistically signific :ant difference between the 12-E and 12-NE animals. A Pearson-Palmer scatter diagram (Fig. 3) plotting treadmill walk ;ing vs dendrite-bundle ratings revealed a correlation coefficient of 0.13. 1This lack of correlation between the two ratings is an argument against the hypothesis that bundle development is essential for alternating flexor- .extensor motor patterns.

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r - 0.133

0.0

1.0

2.0

3.0

TREADMILL

4.0

5.0

6.0

7.0

RATING

FIG. 3. A Pearson-Palmer scatter diagram demonstrates the lack of correlation between walking ability (abscissa) and dendrite bundle complexity of lumbrosacral cord (ordinate). Data from all four groups of spinalized animals are included.

DISCUSSION Much of the literature on development points toward the phenomenon of neuronal interaction as a causal factor in shaping of dendrite trees. Rambn y Cajal (24) reported that during development dendrites of spinal motoneurons undergo a period of growth and duplication followed by another period of reabsorption and regulation. He proposed that the ultimate shape of the dendrite tree was dependent not only on the pattern of axonal afferent fibers but on functional activity as well. More recent work (2, 7, 17, 27, 3 1, 34) supports and extends the correlative relationship between dendritic morphology and presynaptic inputs. The alpha motoneuron receives synaptic terminals from dorsal root afferent fibers, propriospinal and supraspinal tracts, as well as spinal interneurons. Although most of the descending tracts terminate on interneurons in laminae IV to VIII (22, 23) direct monosynaptic connections from supraspinal tracts have been described for the vestibulospinal and reticulospinal tracts in the cat (9, 10). Monosynaptic connections from long propriospinal fibers, though sparse, have also been described (6, 11, 15, 20). Trauma to the cord causes retraction of axonal afferent fibers to alpha motoneurons from both spinal interneurons and supraspinal tracts (5, 14, 18). Monosynaptic connections are not mature at birth or at 2 weeks postnatal (27, 32), so it is not unexpected that the dendrites and dendrite bundle systems which they target do not exhibit normal maturation. The arrest in development and actual deterioration of bundle structure that occurs in the older cordotomized animal might also be expected in light of literature emphasizing the importance of synaptic input in maintenance

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of dendrite arborization (5). It is surprising, however, that compensatory afferent collateralization, which was reported to occur in animals with hemisected cords (7, 13, 14, 18, 19) does not make up for this deficit of information from upper centers in stimulating growth and in maintaining dendritic trees. Because our animals were maintained an average of 4 months postcordotomy, there was ample time for sprouting and collateralization to occur. If sprouting does occur, either it may not be sufficient to stimulate new growth in dendrites that have just undergone a period of retraction, or it may lack specific instruction for directed dendrite growth. Although no positive effect of exercise on development or maintenance of bundles can be demonstrated statistically, other studies suggest that exercise in the developing cat stimulates growth of dendritic trees in the sensory and motor cortices. After repeated use of one forelimb of developing kittens, Jensen and Spinelli (12) found a significant dendritic difference in the cerebral hemisphere contralateral to the exercised limb. A key finding in our study is the lack of correlation between dendritic bundles and forced treadmill walking ability. This tends to argue against the hypothesis that bundle maturation is a prerequisite for alternating flexor-extensor patterns needed in walking. Other workers, though largely confirming the Scheibel’s morphological studies, proposed other functions for the bundles. Matthews et al. (16) suggested that synchronization of neuronal firing might be a more appropriate function based on the closeness of the dendrites involved in the bundles. Anderson (l), in a study of rat spinal cord, found the most complex bundling in the sixth lumbar segment, an area involved with gluteal muscle and knee-flexor innervation. He conjectured that this area of the cord had little to do with alternating flexorextensor movement. It must be pointed out, however, that a hip generator system is thought to be an integral part of the entire spinal generator (3, 9). Although much of the research on spinal walking suggests that the interneuron is of primary importance in the spinal generator (3, 9), the dendrite bundle formation could be essential in postural and environmental adjustments during normal walking. If, indeed, as the data tend to suggest, dendrite bundles are not directly involved in reciprocal limb movements or alternating flexor-extensor patterns, what then is their function? In a review of dendrite bundle literature, Roney et al. (26) suggested a number of possible functions for dendrite bundles including: (i) maintenance of neuronal firing activity over a sufficient time period as a substrate for reentrant activity; (ii) synchronization of input rather than information processing; and (iii) statistical reliability. This is a concept whereby interactive effects of the elements of the dendrite bundle may enhance the reliability of spike response patterns of the involved motoneurons. Other suggested roles more oriented toward higher functions

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of the central nervous system include the provisions of a backup system to prevent stored information from being destroyed by local brain damage, or a reservoir for enhanced memory storage capacity. It is possible that our data might fit into one of these categories rather than the originally postulated hypothesis. REFERENCES 1. ANDERSON, W. J., M. W. STROMBERG, AND E. J. HINSMAN. 1976. Morphological characteristics of dendrite bundles in the lumbar spinal cord of the rat. Brain Res. 110: 215-227. 2. CONRADI, S., AND L. 0. RONNEVI. 1975. Spontaneous elimination of synapses on cat spinal motoneurons after birth: do half of the synapses on the cell bodies disappear? Brain Res. 92: 505-5 10. 3. EDGERTON, V. R., S. GRILLNER, A. SJOSTROM, AND P. ZANNGER. 1976. Central generation of locomotion in vertebrates. Pages 459-464 in R. M. HERMAN, S. GRILLNER, P. S. G. STEIN, AND D. G. STUART, Eds., Neural Control @Locomotion. Plenum, New York. 4. FAYEN, N. A., AND D. VIALA. 1976. Development of locomotor activity in young chronic spinal rabbits. Neurosci. Lett. 3: 329-333. 5. GELFAN, S., G. KAO, AND H. LING. 1972. The dendritic tree of spinal neurons in dogs with experimental hindlimb rigidity. J. Camp. Neurol. 146: 143-17 1. 6. GIOVANELLI-BARILARI, M., AND H. G. J. M. KUYPERS. 1969. Propriospinal fibers interconnecting the spinal enlargements in the cat. Brain Rex 14: 321-330. 7. GOLDBERGER, M., AND M. MURRAY. 1973. Restitution of function and collateral sprouting in the cat spinal cord: the deafferented animal. J. Camp. Neural. 158: 37-54. 8. GRILLNER, S. 1975. Locomotion in vertebrates; central mechanisms and reflex interaction. Physiol.

Rev. 55: 247-307.

9. GRILLNER, S., T. HONGO, AND S. LUND. 1966. Descending pathways with monosynaptic action on motoneurons. Acta Physiol. Stand. Suppl. 277: l-60. 10. GRILLNER, S., AND S. LUND. 1968. The origin of a descending pathway with monosynaptic action or flexor motoneurons. Acta Physiol. Stand. 74: 272-284. 11. JANOWSKA, E., A. LUNDBERG, W. J. ROBERTS, AND D. STUART. 1974. A long propriospinal system with direct effect on motoneurons and/or interneurons in the cat lumbosacral cord. Exp. Brain Res. 21: 169-194. 12. JENSEN, F. E., AND D. N. SPINELLI. 1979. Early experience effect on dendrite bundles. Sot.

Neurosci.

Abstr.

5: 503.

13. LIU, C. N., AND W. W. CHAMBERS. 1958. Intraspinal sprouting of dorsal root axons. Arch.

Neural.

Psychiatr.

79: 46-61.

14. LIU, C. N., AND C. Y. LIU. 1971. Role of afferents in maintenance of dendritic morphology. Anat. Rec. 169: 369. 15. LUND. S., AND 0. POMPEIANO. 1968. Monosynaptic excitation of alpha motor neurons from SUpK+Spind structures in the cat. Acra Physiol. Stand. 73: I-21. 16. MATTHEWS, M. A., W. D. WILLIS, AND V. WILLIAMS. 1971. Dendrite bundles in lamina IX of cat spinal cord: a possible source for electrical interaction between motoneurons? Anat.

Rec. 171: 313-328.

17. MOREST, D. K. 1969. The growth of dendrites in the mammalian brain. Z. Anat. Entwicklungsgesch. 128: 290-3 17. 18. MURRAY, M., AND M. GOLDBERGER. 1973. Restitution of function and collateral sprout-

440

19. 20. 21. 22.

REBACK,

SCHEIBEL,

AND

SMITH

ing in the cat spinal cord: the partially hemisected animal. J. Camp. Neural. 158: 1936. MCCOUCH, G. P., G. M. AUSTIN, C. N. Lru, AND C. Y. Lru. 1957. Sprouting as a cause of spasticity. J. Neurophysiol. 21: 205-316. MCLAUGLIN, B. 1972. Propriospinal and supraspinal projections to the motor nuclei in the cat spinal cord. J. Camp. Neural. 144: 475-500. NESMAYANOVA, T. N. 1977. Experimental Studies in Regeneration ofspinal Neurons. Wiley, New York. NYBERG-HANSON, R. 1966. Functional organization of descending supraspinal fibre systems to the spinal cord. Anatomical observations and physiological correlations. Ergeb. Anat.

Entwicklungsgensch.

39: 1-48.

23. NYBERG-HANSON, R. 1969. Do cat spinal motoneurons receive direct supraspinal fibre connections: a supplementary silver study. Arch. Ital. Biol. 107: 67-78. 24. RAM~N Y CAJAL, S. 1909. Histologie du SysQme Nerveaux de I’Homme et des VertkbrtG, 2 ~01s. Institute Ram6n y Cajal del CSIC, Madrid (L. Azoulay, Transl. 1952-1955). 25. REBACK, P. 1979. The effect of cordotomy on dendrite bundles and treadmill walking in kittens. Sot. Neurosci. Absir. 5: 175. 26. RONEY, K. J., A. B. SCHEIBEL, AND G. L. SHAW. 1979. Dendritic bundles: survey of anatomical experiments and physiological theories. Brain Res. Rev. 1: 225-27 I. 27. RONNEVI, L. O., AND S. CONRADI. 1974. Ultrastructural evidence for spontaneous elimination of synaptic terminals on spinal motoneurons in the kitten. Brain Res. 80: 335359. 28. SCHEIBEL, M. E., AND A. B. SCHEIBEL. 1970. Organization of spinal motoneuron dendritic bundles. Exp. Neurof. 28: 106-l 12. 29. SCHEIBEL, M. E., AND A. B. SCHEIBEL. 1970. Developmental relationships between spinal motoneuron dendritic bundles and patterned activity in the hindlimb of cats. Exp. Neural.

28: 328-335.

30. SHURRAGER, P. S., AND R. A. DYKMAN. 31.

Physiol. Psychol. 44: 252-262. SKOFF, R. P., AND V. HAMBURGER.

1951. Walking spinal carnivores. J. Comp.

1974. Fine structures of axonal growth cones in embryonic chick spinal cord. J. Comp. Neural. 1% 107-148. 32. SKOGLAND, S. 1969. Growth and differentiation with special emphasis on the central nervous system. Ann. Rev. Physiol. 31: 19-42. 33. SMITH, J. L., L. A. SMITH, R. F. ZERNICKE, AND M. G. HOY. 1982. Locomotion in exercised and nonexercised cats cordotomized at two or twelve weeks of age. Exp. Neural. 76: 393-413. 34. VAUGHN, J. E., T. SIMMS, AND M. NAKASHIRA. 1977. A comparison of the early development of exodendritic and axosomatic synapses upon embryonic mouse spinal motor neurons. J. Camp, Neural. 175: 79-100.