Developmental relationship between spinal motoneuron dendrite bundles and patterned activity in the hind limb of cats

EXPERIMENTAL

NEUROLOGY

is,

Developmental Motoneuron

Relationship

Dendrite in the

MADGE Departments University

328-335 (1970)

Bundles Hind

E, SCHEIBEL

of Awtomy of California

Between and

Limb

of

AND ARNOLD

Spinal

Patterned

Activity

Cats B. SCHEIBEL 1

and Psychiatry and Brain Research Institute, Center for the Health Sciences,Los Angeles,

California Received

90024

July

23,197O

The dendrites of spinal motoneurons are organized in bundles in the mature cat and monkey. These bundles are not present at birth but begin to develop toward the end of the 2nd week when the kitten first shows some capability for stepping, walking, and weight bearing in the hind limb. By the 4th or 5th month when the bundles are fully developed, the behavioral repertoire of the limb is complete. Electromyograms taken at similar times show that reciprocal activity between an agonist-antagonist muscle pair is absent at birth, beginning to appear at 12-14 days, and fully developed at 4-5 months. We suggest that the central program responsible for developing appropriate reciprocal activity between the various muscle groups involved in hind limb activity may be generated in the motoneuron dendrite bundles.

Introduction We have recently shown that the majority of rostrocaudal running dendrites of spinal motoneurons in the mature cat and monkey are arranged in tightly packed bundles (12). Each bundle consists of dendrite shafts from neurons of different motor cell columns representing groups of differing, often antagonistic function. We have suggested that these dendrite bundles might constitute subcenters for processing information and for programming spinal motor output. At birth, the motor capabilities of the hind leg of the kitten are limited. Weight bearing is not yet possible and the crawling movements of the limb are awkward and ineffective. Integrated weight bearing, stepping, and walking movements begin to develop between the 10th and 14th day and are usually well advanced by the 3rd to 4th postnatal week. If the motoneuron dendrite bundles are actually concerned with central programing of 1 This study was supported Health Service.

by Grants

NS 01063 and HD

00972, U.S. Public

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the reciprocal activity of flexors and extensors, it would seem reasonable to inquire whether the bundles develop concomitantly with the increasing motor capacity of the limb. In the following study, we have used the Golgi methods together with electromyography to follow the development of kitten lumbosacral neurons and the correlative motor patterns of an agonist-antagonist muscle pair in the hind limb from birth to neurological maturity (4-5 months). Although no firm conclusions can be drawn, the data may be interpreted as suggesting a relationship between the development of bundles and the onset of effective weight bearing, stepping, and walking activities of the hind limb. Methods

This study is based on examination of approximately 650 Golgi stained sections of lumbosacral spinal cord from 70 kittens, aged 1 day through 5 months. Specimens were stained with one of several modifications of the rapid Golgi method and cut in the horizontal sagittal plane. These were compared to and interpreted in the light of approximately 25,000 Golgi stained sections from kitten spinal cord cut in various planes of section and used in previous studies (S-l 1). Nissl- and reduced silver-stained preparations served as controls. Relevant sections were photographed and drawn directly from the microscope. The electromyogram (EMG) was recorded at succesive epochs in an attempt to establish possible functional correlates for the developing dendrite bundles. Kittens were suspended in a canvas sling arranged to support head and body, but allowing almost unrestricted motion of the extremities. Bipolar concentric recording electrodes were inserted subcutaneously over the gastrocnemius and anterior tibia1 muscles of one hind leg and led to an Offner Type T eight-channel electoencephalograph. Each electrode consisted of a 34-gauge stainless steel enamelled wire inserted into a 2-cm long, 27-gauge hypodermic needle, insulated except at the tip. A platform was usually placed under the hind quarters at such a height as to allow some weight bearing by the limb from which we were recording. During the recording runs, a short time constant was selected in order to screen out as much of the slower movement artifacts as possible. Results

Although dendrite bundles appear to be a regular feature in the lumbosacral region of the ventral horn of mature cats and primates (12)) they cannot be seen in the fetal or newborn animal, As evident from Fig. 1, motoneurons in the late fetal cat appear quite mature. However, the dendrite shafts radiate from the cell body without evidence of the sagittal orientation that appears somewhat later. In sections taken at the 1st postnatal

330

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AND

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day, the beginnings of sagittal organization of dendrites are already apparent (Fig. 1). Measurements of the length of 50 rostrocaudally oriented shafts compared with a similar number of transversely arranged branches show that the former are now longer than the latter by an average of

-a

-b

fetal

Id

12d

4m

FIG. 1. Series of drawings of horizontal sections through ventral horn to show development of motoneuron dendrites and of dendrite bundles. Dendrite systems in fetal cord are essentially radiative. At 1 day of age, the beginnings of sagittal

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22%. The pair of EMG traces led from the regions overlying gastrocnemius and anterior tibia1 muscles shows a picture without clear-cut pattern (Fig. 2). Both muscles produce records characterized by low to medium voltage bursts of varying length without discernible eivdence of phasing of activity between the two. At 12 days of age, further lengthening of the dendrite system is apparent, primarily along the axis of the spinal cord. The mean length of dendrites extending in this direction surpassesthat of transversely oriented shafts by a ratio of at least 2 to 1. Furthermore, as Fig. 1 shows, the peripheral portions of small groups of shafts have begun to follow parallel courses in close apposition for distances of 20-100 p. These appear to represent the initial stage of bundle formation. The EMG taken at 12 days still shows a record without obvious organization. However, as indicated in the second set of traces in Fig. 2, there is some hint of alternation in bursts of EMG activity from gastrocnemius and anterior tibia1 muscles, especially in the latter half of this short strip. At 4-5 months of age, the primarily rostrocaudal dendrite orientation seemsfully stated while dendrite bundles are dense, numerous, and well developed. These bundles sometimes maintain their identity for several millimeters while individual dendrites may continue as part of a bundle for distances of 500-700 p. Individual dendrite shafts often run so closely apposed to each other that no spacecan be resolved between them with the optical miscroscope. Interesting in this regard is the fact that electron microscopy shows that motoneuron dendrites are unique among dendrite systems in the spinal cord, of cold-blooded vertebrates at least, for their complete lack of glial sheathing (Stensaas, personal communication). If this should prove equally true in the mammalian forms we are studying, even more dendritic membrane would thus be available for these long appositional types of contacts between shafts, whatever their functional import may be. The EMG at this time (Fig. 2, third set of traces) shows alternating bursts of activity in the gastrocnemius-anterior tibia1 muscle pair during organization are visible. At 12 days, sagittal orientation is well advanced and beginnings of bundle formation at a, b, and c, are visible. At 4 months, the motoneuron system is essentially mature, sagittal orientation of dendrites is predominant, and dendrite bundles at a, b, and c, are well developed and project along the cord for long distances. Neurons A, B, and C contribute dendrites to more than one bundle. Other abbreviations include vm, ventromedial white matter; vl, ventrolateral white matter; and d, small bundle of commissural dendrites which can be seen as early as the 1st postnatal day. Inset figure shows plane of sections and division of the spinal gray matter after Rexed (J. Corrzp. Nerrrol. 96: 415-495, 1962). Drawn from sections of young cat at various ages, stained by rapid Golgi variants. x lL!,

332

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AND

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Gastroc. +k--,. Ant. Tib.u+

EMG

ld ----

12 d

--

4 m.

[ 200 mv

-.-

1 see FIG. 2. Electromyograms from gastrocnemius and anterior tibia1 muscles of kitten taken at 1 day, 12 days, and 4 months. There is no obvious reciprocal activity in the first pair of traces, while there is a suggestion of alternating sequences toward the end of the second pair, and well developed alternation in the third pair of traces. See text for details. Calibration as indicated.

certain types of stepping-weight bearing movements. This does not represent the only pattern generated by these muscles, and reciprocal sequencing of the two is not always apparent in the records. However, this particular type of pattern and its associated step-walk-weight bearing sequenceis of interest because of its possible relation to the dendrite bundles. If the relationship which we postulate exists, a good deal more must be learned about modes of interaction among dendrites, and the information processing capability of such relationships. Discussion

These data indicate that the organization of spinal motoneuron dendrites in bundles

which

appears

to characterize

cats and monkeys (12) does not apparently

begins

to develop

the mature

lumbosacral

cord

in

exist at birth. The structural complex

in the kitten

between

the

10th

and

14th

postnatal day and appears fully developed by the 4th or 5th month of life. Observation of kittens at progressive states following birth shows &at in

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the immediately postnatal phase, they are unable to bear weight on their hind limbs and that all crawling motions are clumsy and relatively ineffective. Such motions seem primarily dependent on the largest proximal muscle masses with little or no reciprocal flexor-extensor activity identifiable at the knee or ankle joint. Electromyographic monitoring of the unpatterned bursts in an agonist-antagonist pair such as gastricnemius-anterior tibia1 supports these observations. The initial appearance of stepping-walking-weight bearing activity in the hind limb toward the end of the second week is reflected in larval evidences of sequencing in the EMG record. This is also the earliest that motoneuron dendrites can be seen developing bundle formations. Reciprocal sequencing of EMG bursts in antagonists appear fully developed at 4-5 months, coeval with final development of the dendrite bundles in the ventral horn. There is still no consenus as to the mechanisms that control the stepping reaction or the reciprocal activities of its constitutent flexor-extensor pairs. Sherrington (13) initially concluded that movement of the limb itself acts as the stimulus for each sequential element of stepping, the relevant excitatory stimuli coming from deep receptors of the limb. Graham Brown (1) noted that alternating activity of flexors and extensors could be elicited following deafferentation, leading him to postulate an intrinsic mechanism in the spinal cord capable of controlling the alternating activities in stepping. Liddell and Sherrington (4) later conceived of the stretch reflex as an adjustment mechanism, modulating a motor rhythm of central origin, similar to that of Brown. On the basis of their recent reconsideration of the problem, Engberg and Lundberg (2) and Lundberg (5) conclude that “ . . . the basic mechanism of the alternating activation of extensors and flexors in stepping is centrally programmed in the spinal cord and not provided exclusively by reflexes.” As substrate for this central mechanism, a system of paired half centers similar to the original notion of Brown (1) has been suggested. Two interneuron pools are postulated, one activating flexor and another, extensor motoneurons, tied together by reciprocal lines of inhibition (3) and modulated by supraspinal mechanisms (5). Actually, the stepping and walking reflexes are much more complex as has been recently stressed by Lundberg (5). Apart from the more obvious reciprocal activation of extensor-flexor pairs, hip movements are out of phase with movements of other joints. This differentiation turns out to be absolutely necessary in advancing the hind limb of the cat. In addition, there are separate components of “yield” occurring in the stance phase and in locomotion where it contributes to the “spring” of the feline gate (5). These phenomena may ultimately rely on differential patterning of alpha and gamma activation, provided in turn by the central program. And each

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of these motor patterns must yield in turn to more urgent, or more complex, motor behavior signalled by supraspinal centers. With the source of programing still unknown, it seems reasonable to evaluate all structural subsystems which might conceivably be involved. We believe that the motoneuron dendrite bundle offers an hitherto unrecognized substrate in which information processing of considerable complexity may occur. Its absence at birth when the motor behavior is minimal, and its development pari passu with the increasing complexity of motor patterning, much of it reciprocal, suggestsa relationship by inference. It is now known that some dendrite systems may develop direct synaptic relations among themselves providing both inhibition and facilitation on adjacent portions of membrane (7). Although there is no evidence of this kind as yet in the ventral horn, there is convincing data on the efficacy of extrasynaptic mechanismsoperating upon adjacent spinal elements (6). On the basis of the evidence we have put forward, and the data available from other neural systems, we suggest that motoneuron dendrite bundles may provide a significant part of the information processing capability in spinal cord and that they may constitute the source of the central program responsible

for reciprocal

activity

of leg muscIe

pairs

in stepping,

walking,

and weight bearing. References 1. BROWN,

T.

G.

1914. On the nature of the fundamental activity of the nervous with an analysis of the conditioning of rhythmic activity in progression, and a theory of the evolution of function in the nervous system. .I. Physiol. Londolt. 48 : 18-46. ENGBERG, I., and A. LUNDBERG. 1969. An electromyographic analysis of muscular activity in the hindlimb of the cat during unrestrained locomotion. Arta Physiol. Stand. 75: 614630. JANKOWSKA, E., M. G. M. JUKES, S. LUND, and A. LUNDBERG. 1967. The effect of dopa on the spinal cord. 5. Reciprocal organization of pathways transmiit’ng excitatory action to alpha motoneurons of flexors and extensors. Acta Physiol. Stand. 70: 369-388. LIDDELL, E. G. T., and C. S. SHERRINGTON. 1924. Reflexes in response to stretch. Proc. Roy. Sot. Ser. B. 96: 212-242. LUNDBERG, A. 1969. “Reflex Control of Stepping,” pp. 542. The Norwegian Academy of Science and Letters, Universitetsforlaget, Oslo. NELSON, P. G. 1966. Interaction between spinal motoneurons of the cat. J. Neurophysiol. 29 : 275437. RALL, W., G. M. SHEPHERD, T. S. REESE, and M. W. BRIGHTMEN. 1966. Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. ,%+I. Newrol. 14: 44-56. SCHEIBEL, M. E., and A. B. SCHEIBEL. 1966. Spinal motoneurons, interneurons, and Renshaw cells. A Golgi study. Arch. Ital. Biol. 104: 328-353. SCHEIBEL, M. E., and A. B. SCHEIBEL. 1966. Terminal axona] patterns in cat spinal cord. I. The lateral cortico-spinal tract. Brain Res. 2: 333-350.

centers together

2.

3.

4. 5. 6. 7.

8. 9.

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M. E., and A. B. SCHEIBEL. 1968. Terminal axonal patterns in cat cord. II. The dorsal horn. Br& Res. 9 : 32-58. SCHEIBEL, M. E., and A. B. SCHEIBEL. 1969. Terminal patterns in cat spinal cord. III. Primary afferent collaterals. Bvabt Rrs. 13: 417-443. SCHEIBEL, M. E., and A. B. SCHEIBEL. 1970. Organization of spinal motoneuron dendrites in bundles. Exi). Nr24rol. 28: 106112. SHERRINGTON, C. S. 1910. Flexion-reflex of the limb, crossed extension reflex, and reflex stepping and standing. J. PI~ysiol. Londot~ 40: 28-121. SCHEIBEL,

spinal

II.

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