Location of sternocleidomastoid and trapezius motoneurons in the cat

Brain Research, 156 (1978) 339 344 6;) Elsevier/North-Holland Biomedical Press

339

Location of sternocleidomastoid and trapezius motoneurons in the cat

S. RAPOPORT The Rockefeller University, New York, N.Y. 10021 (U.S.A.)

(Accepted July 5th, 1978)

The muscles sternocleidomastoid (SCM) and trapezius (TRAP), although both innervated by the spinal accessory nerve (SAN), mediate quite different movements. SCM is a flexor and rotator of the head, while TRAP is primarily an elevator of the scapula 11, and secondarily an extensor and rotator of the head 1,5. SCM and TRAP are also accessory muscles of respiration, but again, in this respect, these two muscles function differently. During labored breathing, TRAP, in synergism with the other dorsal neck muscles, fixes the head, thus enabling SCM to pull the clavicle craniad and to elevate the sternum2, 5,1~,~4. Because of the distinctly different functions of TRAP and SCM, it might be expected that the motoneurons of these two muscles receive different reflex connections and are anatomically segregated into two cell columns. Since SCM is a ventral neck muscle, while T R A P is a dorsal neck muscle, segregation of their respective motoneurons into two distinct cell columns might also be expected from a generalization of principles which govern the organization of other segments of the spinal cordt% 13. Although it is well established that the motor innervation to SCM and TRAP in the cat is carried solely by the SAN 3, and that the somata of motoneurons which give rise to the SAN have been localized to the ventral horn of the C~-C6 segments6-S,l°, 12, this cell column has never been subdivided into its two functional components. The present communication will report upon the results of experiments which demonstrate that the motoneurons of TRAP and SCM are localized to two distinct cell columns in the cervical spinal cord. Two methods were utilized in the localization of SCM and TRAP motoneurons. In one series of experiments the muscles were injected with the enzyme horseradish peroxidase (HRP), and the somata of motoneurons which were labelled by retrograde transport of the HRP were identified on histological sections. In another set of experiments, which were conducted for the purpose of intracellular recording from antidromically identified motoneurons, dye marks were made at locations at which actions potentials or antidromic field potentials from these motoneurons could be recorded. HRP experiments were performed in cats weighing 3.0-3.5 kg. The animals were

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Fig. 1. Identification of SCM and TRAP motoneurons by retrograde transport of HRP injected into the corresponding muscles, a: dark-field photomicrograph of an SCM motoneuron labelled by HRP. Distinct granules and the cell nucleus are demonstrated, b: bright-field photomicrograph of another SCM motoneuron. HRP granules and cell nucleus are distinctly seen. No labelling of the nucleus was noted, c: dark-field photomicrograph of 4 HRP-stained SCM motoneurons carrowsk They are located mediodorsally in the ventral horn of the Ca segment, d : low-power view of a section showing 4 TRAP motoneurons in the left ventral horn. and one SCM motoneuron in the right ventral horn l arrows~. The section was taken at the transition between the C1 and C2 segments. Note that the TRAP motoneurons are located more laterally than the SCM motoneuron, Magnifications: a and b. 518.8: c, × 87.5; d, / 22.5. anesthetized with k e t a m i n e (Vetalar, Parke-Davis) 20 m g / k g i.m. every 30 min. They also received a single injection o f a t r o p i n e sulfate (Lilly) 0.1 mg i.m.. in o r d e r to p r e v e n t aspiration o f respiratory secretions. T h e muscle to be injected l in one cat the left S C M ; in one cat the left T R A P : and in one cat the right S C M and t h e left T R A P ) was identified a n d dissected free o f the s u r r o u n d i n g muscles a n d c o n n e c t i v e tissue. G r e a t care was t a k e n n o t to t r a u m a t i z e the muscle o f interest, or to interfere with its i n n e r v a t i o n or b l o o d supply. A solution o f H R P (Sigma type VI, 40-60 rag/0.3-0.4 ml HzO) was injected at multiple sites a l o n g the muscle, with a small a m o u n t o f e n z y m e deposited at each site. Injections were m a d e until the whole muscle was o b s e r v e d to turn a dark brown color. G r e a t care was t a k e n in o r d e r to a v o i d spillage o f H R P o n t o the s u r r o u n d i n g tissues.

34l After a survival time of 72 h, all cats were anesthetized with sodium pentobarbital (Nembutal, Abbott) 40 mg/kg, and perfused with a paraformaldehyde-gluteraldehyde fixative solution in phosphate buffer 4. Following fixation, 50 or 100 /~m transverse sections of the neuraxis, from mid-pons to C7 spinal segment, were cut on a freezing microtome and reacted with diaminobenzidine and hydrogen peroxide 4. Sections were stained with cresyl violet, and were examined microscopically under bright- and dark-field illumination. Following injection of H R P into the SCM and T R A P muscles, labelled cells, such as the ones illustrated in Fig. 1, were found in the ventral horn of the spinal cord ipsilateral to the injected muscle. As is shown in Fig. la and b, these cells contained a dense concentration of granular H R P reaction product, which filled the cell body exclusive of the nucleus. No evidence was seen of the diffuse type of reaction product thought to indicate direct labelling by the enzyme 9, or of endogenous peroxidase activity t7. Fig. lc and d show the ventral horn at lower magnification, and the arrows indicate the locations of the labelled cells. SCM neurons labelled with H R P were found in the C1 and rostral C2 segments of the spinal cord. Rarely were SCM motoneurons found below the mid-Ce spinal segment. In one experiment in which 260 SCM motoneurons were labelled, all but 8 of the cells were found to lie rostral to the mid-C,) spinal segment. T R A P motoneurons were found from the C1 to the C5 segments of the spinal cord. The largest concentration of T R A P motoneurons was found in the C2-C,~ segments. Thus, although SCM motoneurons are located in spinal segments rostral to mid-C2, T R A P motoneurons are also present in this area, and extend more caudally to at least the C5 segment. Although SCM and T R A P motoneurons overlap longitudinally, they occupy different mediolateral positions in the ventral horn. The SCM motor nucleus occupies a medial and dorsal column in the ventral horn, whereas T R A P motoneurons occupy a more lateral position, bordering on the lateral edge of the ventral horn (Fig. lc and d). As sections of the spinal cord are examined, proceeding from rostral to caudal, the T R A P motor nucleus moves ventrally, so that at Ca it occupies the ventrolateralmost area of the ventral horn (Fig. 2a). Cell volumes of 50 T R A P and 50 SCM motoneurons were estimated using the method described by Schad615. The major (a) and minor (b) axes of the motoneurons, measured through the nucleolus, were determined from camera lucida reconstructions, and applied to the formula 1/6 ~ ab ~/ab, which yields the volume of a non-rotational ellipsoid with third axis ~/ab, multiplied by the correction factor 1.04. The total error in the values obtained by such a procedure has been estimated to be 6 ~o15. For both T R A P and SCM motoneurons the distribution of cell volumes which was obtained best fit a Poisson process. T R A P motoneuron volumes ranged from 2500 to 100,000 cu.#m, with a mean volume of 29,000 (S.D. 170) cu.#m. SCM motoneuron volumes ranged from 4000 to 120,000 cu./~m, with a mean volume of 36,000 (S.D. 190) cu./~m. Thus, while the error involved in determining motoneuron volume is considerable, SCM motoneurons, on the average, are slightly larger than those of TRAP. Electrophysiological experiments were performed in cats weighing from 2.1 to

342

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SCM Cj J

Rostral C2

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Mid_C2

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Fig. 2. a: locations of SCM and TRAP motoneurons, summarized from an experiment in which the right SCM and left TRAP muscles were injected, A total of 260 SCM and 281 TRAP motoneurons were stained in this experiment. Sections are labelled with respect to dorsal root entry zones, b: locations of SCM and TRAP motoneurons, obtained from electrophysiologicalexperiments. Dots in the ventral horn represent dye marks made by ejecting Fast Green dye through the recording pipette during one mediolateral track, and are shown on a camera-lucida reconstruction of the histologic sections. The medial dot marks the located of an SCM motoneuron which was penetrated intracetlulariy. The computer-averaged action potential is drawn on the right (positivity upwards). The lateral dot marks the location at which stimulation of TRAP nerve evoked an antidromic action potential, while stimulation of the SCM nerve was ineffective (positivity upwards). 4.5 kg, which were a n a e s t h e t i z e d with either chloralose (50 mg/kg) or s o d i u m p e n t o b a r b i t a l (30 mg/kg). The S A N b r a n c h e s to S C M and T R A P were dissected a n d m o u n t e d on b i p o l a r p l a t i n u m electrodes for a n t i d r o m i c stimulation. Systematic t r a c k s were m a d e in the C t - C a segments o f the spinal c o r d , with glass micropipettes, while s t i m u l a t i n g the central branches of the nerves to S C M a n d T R A P . T h e r e c o r d i n g eiectrodes were filled with F a s t G r e e n ( F C F ) dye a n d 2 M potassium acetate. T h e dye can be ejected f r o m the pipettes by passing a c a t h o d a l c u r r e n t t h r o u g h them. W h e n 5-20 n A are passed for 5-7 rain. the dye p r o d u c e s a very w e l l - d e l i m i t e d m a r k in the spinal cord. which can be readily identified in frozen sections 16. D u r i n g t r a c k i n g a search was m a d e for single m o t o n e u r o n s , o r for field potentials, which were a c t i v a t e d by a n t i d r o m i c s t i m u l a t i o n o f the muscle nerves The locations of such a n t i d r o m i c a l l y a c t i v a t e d field potentials or m o t o n e u r o n s were m a r k e d , a n d r e c o n s t r u c t e d f r o m histological sections. The o b s e r v a t i o n s m a d e on the H R P e x p e r i m e n t s were c o n f i r m e d by the electrophysiological data. D u r i n g a n t i d r o m i c s t i m u l a t i o n o f the S C M nerve, m o t o n e u r o n s were regularly f o u n d in the Ct and r o s t r a l C2 segments. S e l d o m c o u l d any m o t o n e u r o n s or field potentials be recorded below mid-C2 u p o n a n t i d r o m i c stimul a t i o n o f the S C M nerve. Out of" 35 a n t i d r o m i c a l l y identified S C M m o t o n e u r o n s only one was found in the caudal C2 segment. O n the o t h e r hand, T R A P m o t o n e u r o n s c o u l d be regularly identified in the Ct, Ce a n d Ca spinal segments (no t r a c k i n g was p e r f o r m e d in m o r e c a u d a l segments) o n a n t i d r o m i c s t i m u l a t i o n o f the T R A P nerve.

343 A n t i d r o m i c a l l y identified S C M m o t o n e u r o n s were always f o u n d in a medial a n d dorsal location in the ventral h o r n (Fig. 2b). Immediately ventral to the area where SCM m o t o n e u r o n s could be antidromically activated, and at the same laterality, field potentials and single m o t o n e u r o n s could be recorded u p o n a n t i d r o m i c stimulation of the nerves to biventer cervicis and complexus. In contrast, antidromically identified T R A P m o t o n e u r o n s were always found very laterally in the ventral h o r n (Fig. 2b). At each level which was examined, the medial edge of the T R A P m o t o n e u r o n column was separated by 100 # m or more from the lateral border of the SCM column. In these experiments, antidromically identified splenius m o t o n e u r o n s were f o u n d to occupy an area in the ventral h o r n between T R A P a n d biventer-cervicis-complexus motoneurons. Thus, it is apparent, from both the anatomical a n d electrophysiologic data, that SCM and T R A P m o t o n e u r o n s occupy two distinct cell columns in the spinal cord. It will now be of interest to determine whether there exist different segmental reflex inputs and c o n t r i b u t i o n s from descending tracts to these two m o t o n e u r o n columns. The a u t h o r is pleased to acknowledge the advice and support of Prof. V. J. Wilson. The technical assistance of Ms. S. W o n g is greatly appreciated. This work was supported in part by N1H G r a n t NS 02619.

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