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The lateral cervical nucleus in the cat. I. A Golgi study
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BRAIN RESEARCH
T H E L A T E R A L C E R V I C A L N U C L E U S IN T H E CAT. I. A G O L G I S T U D Y
JAN WESTMAN
Institute of Human Anatomy, University of Uppsala, Uppsala (Sweden) (Accepted March 1lth, 1968)
INTRODUCTION
The lateral cervical nucleus (LCN) in the cat is a longitudinal column of nerve cells in the first and second cervical segments of the spinal cord 3°. it is situated ventrolaterally to the dorsal horn. The L C N was recognized as early as 1899 (ref. 43) but it was then misinterpreted as being a portion of the dorsal horn 28. It was first described as a separate entity by Caja1.5. The nucleus was studied in detail by Rexed2S, z°, Rexed and Brodal zl and Brodal and Rexed 3. With an experimental neuroanatomical technique these authors :~,31 also demonstrated its spinal afferent fibres, ascending in the dorsal part of the lateral funiculus. Using neurophysiological techniques Rexed and StrSm:r~, ~3 showed that the LCN receives cutaneous impulses from ipsilateral nerves, relayed synaptically at a level near the entrance of the afferent nerve fibres into the spinal cord. After these initial studies several investigations of the LCN and its connections were made in cats with different techniques1,'),4, 6 14,16 is,z0 26,35,38,39. These investigations confirmed and extended previous knowledge. The LCN projects to the cerebral cortex6,18, 20-2~,~4 via the ventral posterior nucleus of the thalamus~, 1~,~'. Besides cutaneous afferents it also receives muscle and joint afferents'~3, '~5. This Golgi study is intended as a basis for an ultrastructural investigation which has already been started and partially published 41. The L C N has been described in Golgi studies before~',9-r,. Cajal ,~ stated that the nerve cells had a characteristic appearance. They were mostly spherical with curved 'hairy' dendrites, which remained within the nucleus or its immediate neighbourhood. The axon finally took a vertical course, probably ascending in the lateral funiculus. The afferent fibres were of two kinds, thin fibres--collaterals from the lateral funiculus - - and thick fibres. Both types were regarded by Cajal to enter the nucleus from the dorsal spinocerebellar tract. Ha and Liu 10 12 observed three types of synaptic terminals in the LCN: basket-like calyces, terminal clusters and terminals of passage. It was possible for one single fibre to establish contact with more than one neurone and one neurone was able to receive numerous terminals of different types and from different fibres. Brain Research, l0 (1968) 352-368
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MATERIAL AND METHODS
The upper part of the cervical spinal cord of 94 cats, ranging in age from newborn to adult, were stained according to the rapid Golgi method described by Strong ~7, the Golgi-Kopsch techniquO 5 or the Golgi-Cox method described by Shol136. Thirty-seven animals were 1 week of age or less, 34 were kittens between I week and 3 months old and 23 were adult. After surgical anaesthesia the thorax and the right atrium of the heart were opened for exsanguination of the animal. The upper part of the cervical spinal cord was then dissected out and the first three cervical segments were removed, cut into smaller pieces and immersed in the fixation solution after a short rinsing in physiological salt solution. Sometimes the three segments were kept in one piece. Twenty of the animals died during different kinds of operations on the central nervous system. In these the thorax was opened immediately after death for perfusion or exsanguination of the animal. The fixation solution for the rapid Golgi method was 2 ~o potassium dichromate and 0 . 2 ~ osmium tetraoxide. The specimens were left for 2-6 days in darkness at room temperature. They were then rinsed successively in a solution of 0.75 ~ silver nitrate and finally kept in such a solution for 1-2 days. still in darkness and at room temperature. A total of 68 animals were stained according to the rapid Golgi method. Five of these were perfused with potassium dichromate-osmium tetraoxide solution before impregnation according to a method recently published by Morest and Morest 19. Immersion fixation for the Golgi-Kopsch method was carried out for 24 h with a solution of 2 0 ~ formalin and 2.8% potassium dichromate. After this the specimens were kept in 3.5 ~o potassium dichromate in darkness at room temperature for 3-7 days. The specimens were then transferred to 0.75 ~o silver nitrate and then treated in the same way as the material stained according to the rapid Golgi method. Twelve kittens or adult cats were treated in this way. Five of the animals were perfused with buffered formalin before impregnation according to a modified Golgi-Kopsch method, described by Westrum and Lund4L The fixative in the Golgi-Cox technique was 1.1 ~o potassium dichromate. t. 1 ~o mercuric chloride and 0.5 ~ potassium chromate. Impregnation was continued for about 8 weeks. Fourteen kittens and adult cats were impregnated by this method. Two of these were perfused with the fixative prior to impregnation, the others were immersed immediately. The specimens were always dehydrated in acetone and alcohol and embedded in celloidine before the sectioning, which was performed on a sliding microtome. The specimens impregnated according to the rapid Golgi method were cut at 100-150 #, the Golgi-Kopsch material at 100-150 /~ and the Golgi-Cox specimens at 200 #. Most of the spinal cords were cut in transverse section but different kinds of longitudinal sections were also made. The Golgi-Cox sections were placed in 5 ~ potassium sulphide 0.5-1 h before mounting, as described by Shol136. The sections were mounted under cover glasses in Cedax (Merck, Darmstadt}. Drawings of the preparations were made with a special apparatus (Zeiss binocular Brain Research, 10 (1968) 352-368
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d r a w i n g a p p a r a t u s ) which projects a picture o f the d r a w i n g p a p e r and pencil into the m i c r o s c o p e column. By adjusting m i c r o s c o p e and table illumination it is possible to see p r e p a r a t i o n and d r a w i n g p a p e r and pencil at the same time. P h o t o g r a p h s were made in a Leitz p h o t o m i c r o s c o p e . RESULTS The L C N occupies a position ventrolateral to the dorsal horn in the first and second cervical segments. It is s u r r o u n d e d on all sides by the white matter of the lateral funiculus. Only in the cranial part o f the first cervical segment is it connected with the reticular f o r m a t i o n o f the dorsal horn by strands o f cells. Within the nucleus, fibres passing in every direction can be seen, although those crossing the L C N laterally to medially or vice versa d o m i n a t e in transverse sections. Afferent fibres can be seen entering the L C N from all sides in such sections.
Afferent fibres from the dorsolateral and lateral sMe o[" the L C N These represent the main part o f the afferent fibres to the nucleus or at least
Fig. 1. Rapid Golgi impregnation. Transverse section of spinal cord from'kitten, 7 days old. The dorsal horn is at the upper left. The figure shows an afferent fibre bundle (arrow) to the LCN. 80 , . Fig. 2. Rapid Golgi impregnation. 'Transverse section of spinal cord from kitten, 7 days old. The dorsal horn is at the upper left. The figure shows two afferent fibre bundles (long arrows) to tl',e LCN. The bundle (short arrow) that seems to enter the nuclet, s from the ventrolateral side is the accessory nerve. 80 ×.
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Fig. 3. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 4 days old: Coarse afferent fibre from the lateral side with a large termination field. Key figure above. those which are most easily impregnated by the Golgi method, in such transverse sections that have been extensively impregnated according to the rapid Golgi method, it is possible to see these afferent fibres forming a bundle as they enter the L C N from the dorsolateral side (Fig. 1). Instead of one bundle two may be present, entering the nucleus at different places (Fig. 2J or both converging towards the dorsolateral part of the L C N . A few of these axons are beaded with about 10 # between each bead (Fig. 3~ but most of them are not. The impregnated fibres are mostly rather coarse but some are fine. M o s t of the axons divide dichotomously several times before or after entering the LCN. After dividing, the coarse fibres end with clusters of terminal b o u t o n s and boutons de passage. The terminal fields o f these axons are rather large (Fig. 3) and in specimens with well impregnated fibres a single fibre can be seen covering almost half the nucleus in a transverse section. The fine fibres seem to have a smaller terminal field where b o u t o n s de passage are the most c o m m o n terminals (Fig. 4~ A single L C N neurone seems to receive terminals from more than one afferent fibre and also both types of terminals. Some of the fibres entering the L C N from the dorsolateral side are collaterals from vertical fibres which leave them at right angles. One vertical fibre can give off several collaterals all terminating within the nucleus. Some of the branching fibres can also be followed back to a vertical stem fibre terminating within the LCN. Brain Research. 10 (1968) 352-368
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Fig. 4. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 16 days old. The figure demonstrates the restricted terminal field and the boutons de passage of a fine afferent fibre to the LCN. Key figure to the right, showing the fibre entering the dorsal part of the LCN. Fig. 5. Rapid Golgi impregnation. Kitten, 3 days old. Longitudinal section. The figure shows a coarse fibre descending within the LCN from the lateral side, with coarse beads or boutons de passage. To the left is the cell body of an LCN neurone. Fig. 6. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 7 days old. The figure shows double collaterals entering the LCN from a fibre crossing the nucleus on its ventral side. Key figure above.
In a l o n g i t u d i n a l s e c t i o n o n e v e r y c o a r s e fibre was seen to d e s c e n d w i t h i n the n u c l e u s f r o m t h e lateral side, t e r m i n a t i n g w i t h l a r g e b o u t o n s de p a s s a g e (Fig. 5).
Afferent fibres from the medial side F i b r e s e n t e r i n g the L C N f r o m the m e d i a l side are r a t h e r c o a r s e a n d seem to h a v e t h e s a m e d i s t r i b u t i o n a n d t e r m i n a t i o n as the m a i n p a r t o f the fibres e n t e r i n g the n u c l e u s f r o m t h e l a t e r a l o r d o r s o l a t e r a l side.
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Afferent fibres from the dorsal and ventral side Fibres entering the nucleus from its dorsal and ventral sides also have the same d i s t r i b u t i o n a n d t e r m i n a t i o n as the coarse ones from the lateral or dorsolateral side. One of the afferent fibres from the dorsal side which were studied was a collateral from a vertical fibre. Other fibres entering the L C N from its ventral or dorsal side are collaterals from transverse fibres crossing the nucleus on its ventral or dorsal side. These collaterals can be double (Fig. 6).
Cell bodies A few cells are small but most of the cells of the lateral cervical nucleus are
Fig. 7. Golgi-Kopsch preparation from kitten, 2 months old, showing cell body of LCN neurone. Note the two small projections (arrows) to the upper right. 800 • . Fig. 8. Golgi-Kopsch impregnation from kitten. 16 days old. The figure shows an LCN neurone with spines (arrows) on the primary and secondary dendrites. 800 ~,.
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m e d i u m - s i z e d to large. A n u m b e r o f the cells have a slightly r o u n d e d cell b o d y while others are star- or spindle-shaped. Well i m p r e g n a t e d neurones have 4-9 main dendrites, a n d mostly 6. The axon emerges from the cell b o d y a b o u t twice as often as f r o m the dendrites. Very often the axon leaves with an axon hillock which, however, is often ' h a i r y ' with terminals o f other axons. Very small projections from the cell b o d y have been observed in the G o l g i - K o p s c h material (Fig. 7). A x o n terminals also a p p e a r on the cell b o d y proper. These terminals are sometimes the c o n t i n u a t i o n o f a thin axon having b o u t o n s de passage and winding a r o u n d the dendrites. Dendrites
In specimens well i m p r e g n a t e d a c c o r d i n g to the r a p i d G o l g i m e t h o d the dendri~ic tree i~ richly branched, covering in the best i m p r e g n a t e d neurones a l m o s t
-
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Fig. 9. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with its branches. The smooth axon emerges from the cell body and goes to the left. After about 100/~ it gives off an initial collateral (arrow) that branches and ends within the LCN. Inserted at lower right is a magnification of the square in the main figure showing the terminal branches of the collateral. Inserted at upper left is the key figure. Brain Research,
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G O L G I S T U D Y OF THE L A T E R A L CERVICAL N U C L E U S
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359
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Fig. 10. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with 'hairy' dendrites and smooth axon emerging from the cell body, at lower right {arrow). Key figure at upper left shows the longitudinal distribution of the dendritic tree.
100,000 sq.#. Most of the celt bodies of these nerve cells lie in the middle of the dendritic tree (Figs. 9, 10) but some show some kind of asymmetry (Fig. 11). The distribution of the dendrites is about the same in all planes of the spinal cord. There is, however, a slight tendency to a larger extension in the longitudinal axis of the spinal cord (Fig. 10). In the Golgi specimens most of the dendrites also tend to run longitudinally. The dendrites of the neurones seldom extend beyond the borders of the LCN. Occasionally, however, they do and then the dendrites may even enter into the grey matter of the dorsal horn. lamina V of Rexed 29 (Fig. 10). In the rapid Golgi preparations all the dendrites are richly covered with axon terminals giving them a rather characteristic 'hairy' appearance. The fine dendrites seem to be richer in such terminals than the coarse ones. In successfully impregnated Golgi-Kopsch specimens, spines can be seen projecting from the dendrites in all directions (Fig. 8). Spines can be found on the primary dendrites but they are more numerous on the subsequent ones. N o difference was found in the distribution and number of dendritic spines in cats of different ages. Brain Research, 10 (1968) 352-368
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°
11 Fig. 11. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with the axon emerging from the cell body. The axon ascends and then turns towards the dorsal horn. Note the collateral leaving the axon at right angles.
Axons
The axons, which are thinner than the dendritic main branches, emerge fi'om the cell body or from a dendrite, oftenwith awell defined axon hillock. In most cases terminals of other axons can be found on this hillock. The initial part of the axons can be orientated in various directions running medially as often as laterally, ventrally as often as dorsally, ascending as often as descending, in Golgi Kopsch, GolgiCox and most of the material impregnated according to the rapid Golgi method, only the first part of the axon is impregnated. In one of the perfused spinal cords prepared according to the rapid Golgi method, however, the axons can be followed for several hundred/z. Sections in which this has been found have all been cut longitudinally and obliquely in relation to the median plane. Most of the axons are smooth but some of them have beads starting 30-100/z from the axon hillock. It has not been possible to follow these well impregnated axons to their final destination but in most cases they have been seen turning towards the grey matter of the spinal cord after having ascended or descended or having made a loop in the neighbourhood of the parent cell. One axon has been followed 350/~, entering the grey matter (Fig. 1 I) at a point ventromedial to the LCN. Axon collaterals
In well-impregnated, perfused specimens in which longitudinal sections were Brain Research, I0 (1968) 352-368
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cut obliquely to the median plane, the axons were found to give off collaterals at a distance of 70-130/~ from the cell body. These collaterals are very thin, leaving the axon at right angles (Fig. 11 ). Most of the collaterals branch repeatedly and terminate within the LCN mainly with what seems to be small boutons (Fig. 9, insert) or free endings. In well-impregnated specimens the LCN neurones were surrounded by a delicate network of very fine fibres dividing repeatedly. Sometimes these fibres could be seen starting as collaterals from coarser fibres. These very fine fibres were of the same thickness as the LCN axon collaterals. Relations between different L C N neurones Because of the vast distribution of the dendritic trees and the relatively large number of nerve cells within the LCN - - 2800 to 2950 on each side of the first cervical segment and lower medulla zl - - the dendrites are heavily intermingled with each
loo,u Fig. 12. Rapid Golgi preparation. Transverse section from kitten, 7 days old, The figure demonstrates a typical neurone in the medial part of the LCN. The axon emerges from the cell body and travels medially to enter the grey matter of the spinal cord, Fig. 13. Golgi-Cox impregnation. Transverse section from kitten. 2 months old. The figure shows six neurones in the upper part of the first cervical segment where the LCN is still connected to the dorsal horn. The three lateral cells are LCN neurones, the medial neurone belongs to lamina V (according to Rexed). The two intermediate ones are atypical LCN neurones. Brain Research, 10 (1968) 352-368
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Fig. 14. Rapid Golgi impregnation. Transverse section from kitten, 3 days old. The figure shows a neurone of lamina V (Rexed). The dendrites seem to stop at the border of the LCN or pass tile nucleus.
other and with neighbouring cell bodies. This is best seen in the Golgi Cox specimens. Closely related neurones The main part of the LCN neurones are very characteristic and have the features presented above. Some of the nerve cells in the rostromedial part of the nucleus where it is connected to the dorsal horn are not typical, however. They have long slender dendrites almost without branches. The dendrites sometimes extend beyond the border of the LCN (Figs. 12, 13). These nerve cells show a close resemblance to the nerve cells of the neighbouring lamina V of Rexed '9. Sometimes their axons can be followed into the grey matter of the spinal cord (Fig. 12). The nerve cells of the grey matter of the spinal cord immediately medial to the LCN (within the lateral division of Rexed's lamina V, ref. 29) have very long, sparsely branched dendrites which extend into the neighbouring white matter of the spinal cord. These dendrites seem to avoid the LCN in that they mostly pass beside the nucleus or stop at its border (Fig. 14). Most of the observations on the nerve cells and afferent fibres in the LCN were made in material from kittens impregnated according to the rapid Golgi method. The small dendrites were less extensively impregnated in the older and adult animals than in the very young ones, and correspondingly the axons were only impregnated in their proximal part, if at all, in the older group. Some of the preparations from adult cats made according to the rapid Golgi technique have nevertheless been fairly successful. The results reached on the material from adult cats have confirmed those obtained from kittens. The distribution and branching of the proximal dendrites were the same regardless of age. The spines were studied mainly with the Golgi-Kopsch technique on kittens up to several months of age. The Golgi-Kopsch preparations have sometimes also shown terminal branching of the afferent axons. The relation between the different LCN neurones was studied in Golgi-Cox preparations from kittens and adult cats. Brain Research, 10 (1968) 352-368
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DISCUSSION
Afferentfibres Most of the afferent fibres to the LCN are rather coarse and enter the nucleus from all sides, although those from the dorsal part of the lateral funiculus dominate. These latter fibres branch richly and have large terminal fields in the nucleus. The contacts are probably made with terminal clusters of boutons and with boutons de passage. Sometimes the afferent fibres are collaterals of vertical fibres and sometimes they are ascending stem axons terminating within the nucleus. There are also a fe~ fine afferent fibres which appear to have smaller terminal fields where boutons de passage are the dominating type of terminal. Whether this means that there are really two different afferent systems is difficult to decide from this material. The fine fibres might be branches of coarse fibres leaving their parent fibre at a rather long distance from the LCN. One observation presented here indicates the existence of coarse descending fibres to the nucleus. One afferent system seems to be the initial collaterals described here. These are very thin, and after branching they terminate within the nucleus with what seems to be small boutons or tree endings. The axons passing dorsally and ventrally to the LCN and which are seen giving off collaterals to the nucleus in transverse sections (Fig. 6) might well be axons from cell bodies located above or below the plane ol" section. These initial collaterals will be further discussed later on. Afferent fibres to the LCN have been described by CajaD in Golgi preparations and also by Ha and Liu 9 1'). Cajal described two types of fibres, coarse and thin. The thin fibres were regarded by him as collaterals of the dorsal spinocerebellar tract terminating with boutons and free endings. Ha and Liu reported from their investigation with a modified Golgi-Cox method three types of terminals within the LCN. boutons de passage, boutons in terminal clusters and synaptic terminals in baskets (basket or calyx of Held). One single fibre could establish contact with more than one neurone, and one neurone could receive numerous terminals of different types and from different fibres. 3-his investigation thus supports the earlier ones in most respects. In my opinion. however, it is not possible to state either from this Golgi material or from that of Cajal 5 or Ha and Liu 9,12 from what fibre tracts the collaterals from the dorsal part of the lateral funiculus emerge. They may just as well be collaterals from a specific spinocervical tract s as from tracts ascending beyond the rostral pole of the LCN. It can be stated, however, that at least some of the afferent fibres are ascending ones terminating within the LCN. N o baskets of Held have been seen with the impregnation methods used here. The afferent fibres have been seen entering the LCN not only frcm the dorsolateral, lateral and ventrolateral side but from all sides. Coarse and thin fibres may have different modes of termination. There may exist coarse fibres descending to the LCN. The final evaluation of these last two findings wilt have to await further neuroanatomical or neurophysiological investigations. Brain Research, 10 (1968) 352-368
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The spinal afferent fibres to the LCN, demonstrated with neuroanatomical degeneration methods 3,31 or with neurophysiological techniquesS,]3,'23,26,3'~, 33 ascend in the dorsal part of the lateral funiculus. They come from all levels of the spinal cord below the nucleus. Electron microscopical investigations (Westman, in preparation) show that most of the boutons within the LCN are unchanged after hemisection immediately below the nucleus and thus indicate the presence of afferent fibres from sources other than below the level of the LCN. One possible explanation for this could be the presence of initial axon collaterals from the LCN neurones. This will be discussed later on. Another explanation is the presence of cutaneous afferents from the trigeminal nerve. Such afferents have been reported in a single neurophysiological study 39. Two types of afferent fibres - - coarse and thin - - have been described here. This is of interest referring to neurophysiological investigations s in which two types of afferent inflow to the LCN are described. LCNneurones
Cajal 5 stated that the LCN neurones have a characteristic appearance in that they are mostly spherical with curved 'hairy' dendrites which remain within the nucleus or its immediate neighbourhood. Furthermore, he noted that the axons, after some deviations, take a vertical course to ascend within the lateral funiculus on the same side. Ha and Liu 9,19 reported that the dendrites mostly seem to run longitudinally r~ and sometimes entered the grey matter of the dorsal horn 9. They also described a peculiar branched dendritic process that existed beside ordinary spines and which established contact with other dendrites or the parent cell body r~. In the present investigation, Cajal's findings regarding the dendrites of the nerve cells in the LCN have been fully confirmed. The axons, however, seem to go ventromedially to enter the grey matter of the spinal cord if followed far enough. This may seem to be in disagreement with Cajal's findings but is in accordance with other investigations (Boivie and Grant, in preparation) using the Nauta method. The reason for this may be that Cajal did not follow the axons far enough. The initial part of the axon is sometimes ascending. In order to follow the axon into the grey matter it is necessary to have longitudinal sections cut obliquely in relation to the median plane. Ordinary spines project from all dendrites, especially from the small ones. This has been found in preparations treated according to the Golgi-Kopsch method, which is considered to be the best one for demonstrating spines. Small projections, without peripheral swellings, can occasionally be seen to emerge from the cell body. in ultrastructural investigations of the L C N 40,41 spines can be demonstrated on dendrites. Other processes similar to those described here seem to emerge from the cell body. Branched spines from the LCN dendrites or the cell bodies have been demonstrated neither with the three modifications of the Golgi method used here, including some perfused specimens, nor with the electron microscope in serial sections 40. One Brain Research, 10 (1968) 352-368
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can therefore not exclude that the micrographs published by Ha and Liu te show axon terminals on LCN neurones. No differences between cats of different ages regarding the dendritic tree have been observed in this study, although such differences have been reported from studies on the cerebral cortex by the Scheibels 34. This development of neurones in young animals might be a phenomenon characteristic of higher brain centres. A description of the LCN neurones based on frozen sections prepared according to the Glees method by Brodal and Rexed a has shown that the cell processes can be 6-8 on each cell. The processes tend to take a longitudinal course within the LCN and are confined to the nucleus. Ha and l_iuv-' also observed the predominantly longitudinal direction of the LCN dendrites and reported in another study 9 that the dendrites could enter the grey matter of the dorsal horn. The present study confirms earlier ones concerning the direction of the LCN dendrites and shows, in accordance with Ha and Liu 9, that the LCN dendrites occasionally enter the grey matter of the neighbouring dorsal horn. The number of cell processes observed in well-impregnated specimens seems to be the same in this investigation as in the one by Brodal and Rexed 3. Earlier Golgi investigations have failed to demonstrate axon collaterals from the nerve cells in the LCN. The reason for this might be their extreme thinness or the fact that immersion fixation and not perfusion was used by earlier investigators of this region. The orientation of the block at the sectioning is another factor which might have affected the earlier negative findings. Some of the axon collaterals seem to end with terminal boutons. 3"he collaterals of motor cell axons in the ventral horn of near-term cat foetuses, on the other hand, end without terminal boutons as demonstrated by Prestige e7. The presence of initial axon collaterals in the LCN fits in well with what is known about the functional organization of the nucleus. Neurophysiologicat studies have shown recurrent inhibition in the LCN alter antidromic s,l~ as well as orthodromic stimulation 13. These findings indicate the presence of internuncial neurones within the nucleus, activated by boutons from recurrent initial collaterals. This would mean that at least three groups of boutons may be present in the nucleus, one from the fibres ascending in the dorsal part of the lateral funiculus, another from the recurrent axon collaterals, and a third from the supposed internuncial neurones. Whether the terminals from the recurrent collaterals and from the internuncial neurones represent all the unchanged boutons in the electron microscopical material 41 after lesions immediately below the LCN, cannot be decided yet. Experimental neuroanatommal investigations (Westman, in preparation) may show whether the LCN receives descending fibres from other levels of the central nervous system. Some of the cells in the medial upper part of the LCN are atypical and resemble the nerve cells in the neighbouring lateral portion of Rexed's lamina V of the dorsal horn. Here the LCN is often connected with the dorsal horn by small strands of cells. as observed already by Rexed a°. While this investigation was being made, perfusion methods for Golgi-Kopsch ~z and for the rapid Golgi method 19 were published. These methods were adopted for Brain Research.
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the last p r e p a r a t i o n s in this study. In addition, two o f the kittens in the G o l g i - C o x material were perfused with the same solution that was later used for i m p r e g n a t i o n . A difference between the immersed and the perfused material was observed only with the r a p i d G o l g i specimens. Thus axon collaterals were d e m o n s t r a t e d exclusively in perfused specimens, although only a small p a r t o f the p r e p a r a t i o n s (5 out o f 68) were perfused. W h e t h e r this means that perfusion is definitely superior to immersion fixation is not possible to decide, especially when the h a z a r d o u s outcome o f the Golgi i m p r e g n a t i o n is considered.
SUMMARY
The afferent fibres to the lateral cervical nucleus ( L C N ) and its nerve cells were studied in Golgi p r e p a r a t i o n s from 94 cats ranging in age from n e w b o r n to adult. The rapid Golgi m e t h o d , the G o l g i - K o p s c h technique and the G o l g i - C o x m e t h o d were used. Some o f the spinal cords were fixed by perfusion. M o s t o f the afferents to the L C N were observed to come from the dorsal p a r t of the lateral funiculus. Some o f them u e r e ascending fibres t e r m i n a t i n g in the nucleus, others were collaterals o f longitudinally oriented fibres, q-he dendrites and their spines are described and also the axons. The latter were found to give off initial collaterals which t e r m i n a t e within the nucleus. Some o f the cells in the medial u p p e r part o f the L C N were atypical and resembled the cells o f the n e i g h b o u r i n g lateral p o r t i o n o f lamina V o f Rexed o f the spinal cord, rather than the other L C N neurones. The findings are discussed in relation to the n o r m a l ultrastructure of the LCN, to some experimental n e u r o a n a t o m i c a l light- and electron microscope data, and to some physiological findings.
REFERENCES I ANDERSSON, S. A., Projection of different spinal pathways to the second somatic sensory area in cat, Acta physiol, stand., 56, Suppl. 194 (1962) 1-74. 2 BEUSEKOM,G. T. VAN, Fibre Analysis o f the Anterior and Lateral Funiculi o f the Cord in the Cat, IJdo, Leiden, 1955, pp. 82 and 84. 3 Br~ODAL,A., AND REXEO, B., Spinal afferents to the lateral cervical nucleus in the cat. An experimental study, J. comp. Neurol., 98 (1953) 179 212. 4 Buscr~, H. F. M., An Anatomical Analysis o[ the White Matter in the Brain Stem o f the Cat, Van Gorcum, Assen, 1961, pp. 56-57, 59-61, 63. 5 CAJAL,S. RAMdNY, Histologie du SystOme Nerveux de I'Homme et des Vertdbrds, Tome 1, Consejo Superior de lnvestigaciones Cientificas Madrid, 1952, p. 908-91 l (reprint of orig. ed. 1909). 6 CATALANO, J. VON, AND LAMARCHE, G., Central pathway for cutaneous impulses in the cat, Amer. J. Physiol., 189 (1957) 141-144. 7 DrBIAGJO, F., AND GRUNDFEST, H., Afferent relations of inferior olivary nucleus. IV. Lateral cervical nucleus as site of final relay to inferior olive in cat, J. Neurophysiol., 19 (1956) 10-20. 8 GORDON, G., AND JUKES, M. G. M., An investigation of cells in the lateral cervical nucleus of the cat which respond to stimulation of the skin, J. Physiol. (Lond.), 169 (1963) 28-29P. Brain Research, 10 (1968) 352-368
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9 HA, H., AND LIU, C . - N . , A n anatomical investigation on the lateral cervical nucleus of the cat, Anat. Rec., 139 (1961) 234. 10 HA, H., AND LIU, C.-N., Spinal afferents to lateral cervical nucleus and their terminals, Anat. Rec., 142 (1962) 237-238. I 1 HA, H., AND LIu, C.-N., Synaptology of spinal afferents in the lateral cervical nucleus of the cat. Exp. Neurol., 8 (1963) 318-327. 12 HA, H., AND LIU, C.-N., Organization of the spino-cervico-thalamic system, J. comp. Neto'ol.. 127 (l 966) 445-469. 13 HORROBXN, D. F., The lateral cervical nucleus of the cat; an electrophysiological study, Quart. J. exp. Physiol., 51 (1966) 351-371. 14 KRIEGER, H., AND GRUNDFEST, H., Afferent relations of inferior olivary nucleus. Ill. Electrophysiological demonstration of a second relay in dorsal spino-olivary pathway in cat, J. Neurophysiol., 19 (1956) 3 9. 15 KOPSCH, F., Erfahrungen fiber die Verwendung des Formaldehyds bei der Chromsilberimpregnation, Anat. Anz., 11 (1896) 727-729. 16 LANDGREN, S., NORDVALL, A., AND WENGSTR(JM, C., The location of the thalamic relay in the spino-cervico-lemniscal path, Acta physiol, scand., 65 (1965) 164-175. 17 LUNDBERG, A., AND OSCARSSON, O., Three ascending spinal pathways in the dorsal part of the lateral funiculus, Acta physiol, scand., 51 (1961 ) 1-16. 18 MARK, R. F., AND STE1NER, J., Cortical projection of impulses in myelinated cutaneous afferent nerve fibres of the cat, J. Physiol. (Lond.), 142 (1958) 544-562. 19 MOREST,D. K., AND MOREST, R. R., Perfusion-fixation & t h e brain with chrome-osmium solutions for the rapid Golgi method, Amer. J, Anat., 118 (1966) 811-831. 20 MORIN, F., A new spinal pathway for cutaneous impulses, Amer. J. Physiol., 183 (1955) 245-252. 21 MOR1N, F., AND CATALANO, J. VON, Central connections of a cervical nucleus (nucleus cervicalis lateralis of the cat), J. comp. Neurol., 103 (1955) 17-32. 22 MORIN, F., AND THOMAS, L. M., Spinothalamic fibers and tactile pathways in cat, Anat. Rec., 12l (1955) 344. 23 MORIN, F., K1TAI, S. T., PORTNOY, H., AND DERMIJIAN, C., Afferent projections to the lateral cervical nucleus: a microelectrode study, Amer. J. Physiol., 204 (1963) 667~672. 24 NORRSELL, U., AND VOORHOEVE, P., Tactile pathways from the hindlimb to the cerebral cortex in cat, Acta physiol, scand., 54 (1962) 9--17. 25 NORRSELL, U., AND WOLPOW, E. R., An evoked potential study of different pathways from the hindlimb to the somatosensory areas in the cat, Acta physiol, stand., 66 (1966) 19-31. 26 OSWALDO-CRuz, E., AND KIDD, C., Functional properties of neurons in the lateral cervical nucleus of the cat, J. Neurophysiol., 27 (1964) 1-14. 27 PRESTIGE,M. C.,Initiat collaterals of motor axons within the spinal cord of the cat, J. comp. Neurol., 126 (1966) 123-135. 28 REXED, B., The nucleus cervicalis lateratis, a spinocerebellar relay nucleus, Acta physiol, scand., 25, Suppl. 89 (1951) 67 68. 29 REXEO, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 96 (1952) 415-495. 30 REXED, B., A cytoarchitectonic atlas of the spinal cord in the cat, J. comp. Neurol., 100 (1954) 297-379. 31 REXED, B., AND BRODAL, A., The nucleus cervicalis tateralis: a spino-cerebellar relay nucleus, J. Neurophysiol., 14 (1951) 399-407. 32 REXED, B., AND STROM, G., Afferent nervouspathways from forelimb to lateral cervical nucleus, Actaphysiol. scand., 25, Suppl. 89 (1951) 69 70. 33 REXED, B., ANt) STROM, G., Afferent nervous connexions of the lateral cervical nucleus, Acta physiol, scand., 25 (1952) 219-229. 34 SCHEmEL, M., ANt) SCHEmEL, A., Some structural and funtional substrates of development in young cats. In W. A. HIMWlCH AND H. E. HIMWlCH (Eds.), The Developing Brain, Progress in Brain Research, Vol. 9, Elsevier, Amsterdam, 1964, pp. 6-25. 35 SEKt, Y., Some aspects of the comparative anatomy of the spinal cord, Rec. Adv. Res. herr. Syst. (Tokyo), 6 (1962) 908-924. 36 SHOLL, D. A., Dendritic organization in the neurons of the visual and motor cortices of the cat, J. Anat. (Lond.), 87 (1953) 387-406. 37 STRONG, O., Review of the Golgi method, J. comp. Neurol., 6 (1896) 101-127.
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38 TAUB, A., AND BISHOP, P. O., The spinocervical tract: Dorsal column linkage, conduction velocity, primary afferent spectrum, Exp. Neurol., 13 (1965) 1 21. 39 WALL, P. D., AND TAUB, A., Four aspects of trigeminal nucleus and a paradox, J. Neurophysiol., 25 (1962) 110-126. 40 WESTMAN, J., The lateral cervical nucleus in the cat. II. An electron microscopical study of the normal structure, Brain Research, 11 (1968) in press. 41 WESTMAN, J., AND GRANT, G., Electron microscopy of the lateral cervical nucleus in the cat, Acta Soc. Med. upsalien., 5-6 (1965) 259 262. 42 WESTRUM,L. E., AND LUND, R. O., Formalin perfusion for correlative light- and electronmicroscopical studies of the nervous system, J. Cell Sei., 1 (1966) 229-238. 43 ZIEHEN, TH., Makroskopischen und mikroskopischen Anatomie des Riickenmarcks und des Gehirns. In v. Bardelebens Handbuch der Anatomie des Menschen, Vol. 4, I, Jena, 1899, pp. 34 35.
Brain Research, 10 (1968) 352-368
BRAIN RESEARCH
T H E L A T E R A L C E R V I C A L N U C L E U S IN T H E CAT. I. A G O L G I S T U D Y
JAN WESTMAN
Institute of Human Anatomy, University of Uppsala, Uppsala (Sweden) (Accepted March 1lth, 1968)
INTRODUCTION
The lateral cervical nucleus (LCN) in the cat is a longitudinal column of nerve cells in the first and second cervical segments of the spinal cord 3°. it is situated ventrolaterally to the dorsal horn. The L C N was recognized as early as 1899 (ref. 43) but it was then misinterpreted as being a portion of the dorsal horn 28. It was first described as a separate entity by Caja1.5. The nucleus was studied in detail by Rexed2S, z°, Rexed and Brodal zl and Brodal and Rexed 3. With an experimental neuroanatomical technique these authors :~,31 also demonstrated its spinal afferent fibres, ascending in the dorsal part of the lateral funiculus. Using neurophysiological techniques Rexed and StrSm:r~, ~3 showed that the LCN receives cutaneous impulses from ipsilateral nerves, relayed synaptically at a level near the entrance of the afferent nerve fibres into the spinal cord. After these initial studies several investigations of the LCN and its connections were made in cats with different techniques1,'),4, 6 14,16 is,z0 26,35,38,39. These investigations confirmed and extended previous knowledge. The LCN projects to the cerebral cortex6,18, 20-2~,~4 via the ventral posterior nucleus of the thalamus~, 1~,~'. Besides cutaneous afferents it also receives muscle and joint afferents'~3, '~5. This Golgi study is intended as a basis for an ultrastructural investigation which has already been started and partially published 41. The L C N has been described in Golgi studies before~',9-r,. Cajal ,~ stated that the nerve cells had a characteristic appearance. They were mostly spherical with curved 'hairy' dendrites, which remained within the nucleus or its immediate neighbourhood. The axon finally took a vertical course, probably ascending in the lateral funiculus. The afferent fibres were of two kinds, thin fibres--collaterals from the lateral funiculus - - and thick fibres. Both types were regarded by Cajal to enter the nucleus from the dorsal spinocerebellar tract. Ha and Liu 10 12 observed three types of synaptic terminals in the LCN: basket-like calyces, terminal clusters and terminals of passage. It was possible for one single fibre to establish contact with more than one neurone and one neurone was able to receive numerous terminals of different types and from different fibres. Brain Research, l0 (1968) 352-368
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MATERIAL AND METHODS
The upper part of the cervical spinal cord of 94 cats, ranging in age from newborn to adult, were stained according to the rapid Golgi method described by Strong ~7, the Golgi-Kopsch techniquO 5 or the Golgi-Cox method described by Shol136. Thirty-seven animals were 1 week of age or less, 34 were kittens between I week and 3 months old and 23 were adult. After surgical anaesthesia the thorax and the right atrium of the heart were opened for exsanguination of the animal. The upper part of the cervical spinal cord was then dissected out and the first three cervical segments were removed, cut into smaller pieces and immersed in the fixation solution after a short rinsing in physiological salt solution. Sometimes the three segments were kept in one piece. Twenty of the animals died during different kinds of operations on the central nervous system. In these the thorax was opened immediately after death for perfusion or exsanguination of the animal. The fixation solution for the rapid Golgi method was 2 ~o potassium dichromate and 0 . 2 ~ osmium tetraoxide. The specimens were left for 2-6 days in darkness at room temperature. They were then rinsed successively in a solution of 0.75 ~ silver nitrate and finally kept in such a solution for 1-2 days. still in darkness and at room temperature. A total of 68 animals were stained according to the rapid Golgi method. Five of these were perfused with potassium dichromate-osmium tetraoxide solution before impregnation according to a method recently published by Morest and Morest 19. Immersion fixation for the Golgi-Kopsch method was carried out for 24 h with a solution of 2 0 ~ formalin and 2.8% potassium dichromate. After this the specimens were kept in 3.5 ~o potassium dichromate in darkness at room temperature for 3-7 days. The specimens were then transferred to 0.75 ~o silver nitrate and then treated in the same way as the material stained according to the rapid Golgi method. Twelve kittens or adult cats were treated in this way. Five of the animals were perfused with buffered formalin before impregnation according to a modified Golgi-Kopsch method, described by Westrum and Lund4L The fixative in the Golgi-Cox technique was 1.1 ~o potassium dichromate. t. 1 ~o mercuric chloride and 0.5 ~ potassium chromate. Impregnation was continued for about 8 weeks. Fourteen kittens and adult cats were impregnated by this method. Two of these were perfused with the fixative prior to impregnation, the others were immersed immediately. The specimens were always dehydrated in acetone and alcohol and embedded in celloidine before the sectioning, which was performed on a sliding microtome. The specimens impregnated according to the rapid Golgi method were cut at 100-150 #, the Golgi-Kopsch material at 100-150 /~ and the Golgi-Cox specimens at 200 #. Most of the spinal cords were cut in transverse section but different kinds of longitudinal sections were also made. The Golgi-Cox sections were placed in 5 ~ potassium sulphide 0.5-1 h before mounting, as described by Shol136. The sections were mounted under cover glasses in Cedax (Merck, Darmstadt}. Drawings of the preparations were made with a special apparatus (Zeiss binocular Brain Research, 10 (1968) 352-368
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d r a w i n g a p p a r a t u s ) which projects a picture o f the d r a w i n g p a p e r and pencil into the m i c r o s c o p e column. By adjusting m i c r o s c o p e and table illumination it is possible to see p r e p a r a t i o n and d r a w i n g p a p e r and pencil at the same time. P h o t o g r a p h s were made in a Leitz p h o t o m i c r o s c o p e . RESULTS The L C N occupies a position ventrolateral to the dorsal horn in the first and second cervical segments. It is s u r r o u n d e d on all sides by the white matter of the lateral funiculus. Only in the cranial part o f the first cervical segment is it connected with the reticular f o r m a t i o n o f the dorsal horn by strands o f cells. Within the nucleus, fibres passing in every direction can be seen, although those crossing the L C N laterally to medially or vice versa d o m i n a t e in transverse sections. Afferent fibres can be seen entering the L C N from all sides in such sections.
Afferent fibres from the dorsolateral and lateral sMe o[" the L C N These represent the main part o f the afferent fibres to the nucleus or at least
Fig. 1. Rapid Golgi impregnation. Transverse section of spinal cord from'kitten, 7 days old. The dorsal horn is at the upper left. The figure shows an afferent fibre bundle (arrow) to the LCN. 80 , . Fig. 2. Rapid Golgi impregnation. 'Transverse section of spinal cord from kitten, 7 days old. The dorsal horn is at the upper left. The figure shows two afferent fibre bundles (long arrows) to tl',e LCN. The bundle (short arrow) that seems to enter the nuclet, s from the ventrolateral side is the accessory nerve. 80 ×.
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10o,q
3
Fig. 3. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 4 days old: Coarse afferent fibre from the lateral side with a large termination field. Key figure above. those which are most easily impregnated by the Golgi method, in such transverse sections that have been extensively impregnated according to the rapid Golgi method, it is possible to see these afferent fibres forming a bundle as they enter the L C N from the dorsolateral side (Fig. 1). Instead of one bundle two may be present, entering the nucleus at different places (Fig. 2J or both converging towards the dorsolateral part of the L C N . A few of these axons are beaded with about 10 # between each bead (Fig. 3~ but most of them are not. The impregnated fibres are mostly rather coarse but some are fine. M o s t of the axons divide dichotomously several times before or after entering the LCN. After dividing, the coarse fibres end with clusters of terminal b o u t o n s and boutons de passage. The terminal fields o f these axons are rather large (Fig. 3) and in specimens with well impregnated fibres a single fibre can be seen covering almost half the nucleus in a transverse section. The fine fibres seem to have a smaller terminal field where b o u t o n s de passage are the most c o m m o n terminals (Fig. 4~ A single L C N neurone seems to receive terminals from more than one afferent fibre and also both types of terminals. Some of the fibres entering the L C N from the dorsolateral side are collaterals from vertical fibres which leave them at right angles. One vertical fibre can give off several collaterals all terminating within the nucleus. Some of the branching fibres can also be followed back to a vertical stem fibre terminating within the LCN. Brain Research. 10 (1968) 352-368
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IOp.
I00/u 6
Fig. 4. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 16 days old. The figure demonstrates the restricted terminal field and the boutons de passage of a fine afferent fibre to the LCN. Key figure to the right, showing the fibre entering the dorsal part of the LCN. Fig. 5. Rapid Golgi impregnation. Kitten, 3 days old. Longitudinal section. The figure shows a coarse fibre descending within the LCN from the lateral side, with coarse beads or boutons de passage. To the left is the cell body of an LCN neurone. Fig. 6. Rapid Golgi impregnation. Transverse section of spinal cord from kitten, 7 days old. The figure shows double collaterals entering the LCN from a fibre crossing the nucleus on its ventral side. Key figure above.
In a l o n g i t u d i n a l s e c t i o n o n e v e r y c o a r s e fibre was seen to d e s c e n d w i t h i n the n u c l e u s f r o m t h e lateral side, t e r m i n a t i n g w i t h l a r g e b o u t o n s de p a s s a g e (Fig. 5).
Afferent fibres from the medial side F i b r e s e n t e r i n g the L C N f r o m the m e d i a l side are r a t h e r c o a r s e a n d seem to h a v e t h e s a m e d i s t r i b u t i o n a n d t e r m i n a t i o n as the m a i n p a r t o f the fibres e n t e r i n g the n u c l e u s f r o m t h e l a t e r a l o r d o r s o l a t e r a l side.
Brain Research, 10 (1968) 352-368
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35"]
Afferent fibres from the dorsal and ventral side Fibres entering the nucleus from its dorsal and ventral sides also have the same d i s t r i b u t i o n a n d t e r m i n a t i o n as the coarse ones from the lateral or dorsolateral side. One of the afferent fibres from the dorsal side which were studied was a collateral from a vertical fibre. Other fibres entering the L C N from its ventral or dorsal side are collaterals from transverse fibres crossing the nucleus on its ventral or dorsal side. These collaterals can be double (Fig. 6).
Cell bodies A few cells are small but most of the cells of the lateral cervical nucleus are
Fig. 7. Golgi-Kopsch preparation from kitten, 2 months old, showing cell body of LCN neurone. Note the two small projections (arrows) to the upper right. 800 • . Fig. 8. Golgi-Kopsch impregnation from kitten. 16 days old. The figure shows an LCN neurone with spines (arrows) on the primary and secondary dendrites. 800 ~,.
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m e d i u m - s i z e d to large. A n u m b e r o f the cells have a slightly r o u n d e d cell b o d y while others are star- or spindle-shaped. Well i m p r e g n a t e d neurones have 4-9 main dendrites, a n d mostly 6. The axon emerges from the cell b o d y a b o u t twice as often as f r o m the dendrites. Very often the axon leaves with an axon hillock which, however, is often ' h a i r y ' with terminals o f other axons. Very small projections from the cell b o d y have been observed in the G o l g i - K o p s c h material (Fig. 7). A x o n terminals also a p p e a r on the cell b o d y proper. These terminals are sometimes the c o n t i n u a t i o n o f a thin axon having b o u t o n s de passage and winding a r o u n d the dendrites. Dendrites
In specimens well i m p r e g n a t e d a c c o r d i n g to the r a p i d G o l g i m e t h o d the dendri~ic tree i~ richly branched, covering in the best i m p r e g n a t e d neurones a l m o s t
-
i.......................i
loo p
Fig. 9. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with its branches. The smooth axon emerges from the cell body and goes to the left. After about 100/~ it gives off an initial collateral (arrow) that branches and ends within the LCN. Inserted at lower right is a magnification of the square in the main figure showing the terminal branches of the collateral. Inserted at upper left is the key figure. Brain Research,
10 (1968) 352-368
G O L G I S T U D Y OF THE L A T E R A L CERVICAL N U C L E U S
:z=--
359
--*
}o
l0
Fig. 10. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with 'hairy' dendrites and smooth axon emerging from the cell body, at lower right {arrow). Key figure at upper left shows the longitudinal distribution of the dendritic tree.
100,000 sq.#. Most of the celt bodies of these nerve cells lie in the middle of the dendritic tree (Figs. 9, 10) but some show some kind of asymmetry (Fig. 11). The distribution of the dendrites is about the same in all planes of the spinal cord. There is, however, a slight tendency to a larger extension in the longitudinal axis of the spinal cord (Fig. 10). In the Golgi specimens most of the dendrites also tend to run longitudinally. The dendrites of the neurones seldom extend beyond the borders of the LCN. Occasionally, however, they do and then the dendrites may even enter into the grey matter of the dorsal horn. lamina V of Rexed 29 (Fig. 10). In the rapid Golgi preparations all the dendrites are richly covered with axon terminals giving them a rather characteristic 'hairy' appearance. The fine dendrites seem to be richer in such terminals than the coarse ones. In successfully impregnated Golgi-Kopsch specimens, spines can be seen projecting from the dendrites in all directions (Fig. 8). Spines can be found on the primary dendrites but they are more numerous on the subsequent ones. N o difference was found in the distribution and number of dendritic spines in cats of different ages. Brain Research, 10 (1968) 352-368
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°
11 Fig. 11. Rapid Golgi impregnation from kitten, 7 days old. Longitudinal section showing LCN neurone with the axon emerging from the cell body. The axon ascends and then turns towards the dorsal horn. Note the collateral leaving the axon at right angles.
Axons
The axons, which are thinner than the dendritic main branches, emerge fi'om the cell body or from a dendrite, oftenwith awell defined axon hillock. In most cases terminals of other axons can be found on this hillock. The initial part of the axons can be orientated in various directions running medially as often as laterally, ventrally as often as dorsally, ascending as often as descending, in Golgi Kopsch, GolgiCox and most of the material impregnated according to the rapid Golgi method, only the first part of the axon is impregnated. In one of the perfused spinal cords prepared according to the rapid Golgi method, however, the axons can be followed for several hundred/z. Sections in which this has been found have all been cut longitudinally and obliquely in relation to the median plane. Most of the axons are smooth but some of them have beads starting 30-100/z from the axon hillock. It has not been possible to follow these well impregnated axons to their final destination but in most cases they have been seen turning towards the grey matter of the spinal cord after having ascended or descended or having made a loop in the neighbourhood of the parent cell. One axon has been followed 350/~, entering the grey matter (Fig. 1 I) at a point ventromedial to the LCN. Axon collaterals
In well-impregnated, perfused specimens in which longitudinal sections were Brain Research, I0 (1968) 352-368
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cut obliquely to the median plane, the axons were found to give off collaterals at a distance of 70-130/~ from the cell body. These collaterals are very thin, leaving the axon at right angles (Fig. 11 ). Most of the collaterals branch repeatedly and terminate within the LCN mainly with what seems to be small boutons (Fig. 9, insert) or free endings. In well-impregnated specimens the LCN neurones were surrounded by a delicate network of very fine fibres dividing repeatedly. Sometimes these fibres could be seen starting as collaterals from coarser fibres. These very fine fibres were of the same thickness as the LCN axon collaterals. Relations between different L C N neurones Because of the vast distribution of the dendritic trees and the relatively large number of nerve cells within the LCN - - 2800 to 2950 on each side of the first cervical segment and lower medulla zl - - the dendrites are heavily intermingled with each
loo,u Fig. 12. Rapid Golgi preparation. Transverse section from kitten, 7 days old, The figure demonstrates a typical neurone in the medial part of the LCN. The axon emerges from the cell body and travels medially to enter the grey matter of the spinal cord, Fig. 13. Golgi-Cox impregnation. Transverse section from kitten. 2 months old. The figure shows six neurones in the upper part of the first cervical segment where the LCN is still connected to the dorsal horn. The three lateral cells are LCN neurones, the medial neurone belongs to lamina V (according to Rexed). The two intermediate ones are atypical LCN neurones. Brain Research, 10 (1968) 352-368
362
3. WESTMAN
Fig. 14. Rapid Golgi impregnation. Transverse section from kitten, 3 days old. The figure shows a neurone of lamina V (Rexed). The dendrites seem to stop at the border of the LCN or pass tile nucleus.
other and with neighbouring cell bodies. This is best seen in the Golgi Cox specimens. Closely related neurones The main part of the LCN neurones are very characteristic and have the features presented above. Some of the nerve cells in the rostromedial part of the nucleus where it is connected to the dorsal horn are not typical, however. They have long slender dendrites almost without branches. The dendrites sometimes extend beyond the border of the LCN (Figs. 12, 13). These nerve cells show a close resemblance to the nerve cells of the neighbouring lamina V of Rexed '9. Sometimes their axons can be followed into the grey matter of the spinal cord (Fig. 12). The nerve cells of the grey matter of the spinal cord immediately medial to the LCN (within the lateral division of Rexed's lamina V, ref. 29) have very long, sparsely branched dendrites which extend into the neighbouring white matter of the spinal cord. These dendrites seem to avoid the LCN in that they mostly pass beside the nucleus or stop at its border (Fig. 14). Most of the observations on the nerve cells and afferent fibres in the LCN were made in material from kittens impregnated according to the rapid Golgi method. The small dendrites were less extensively impregnated in the older and adult animals than in the very young ones, and correspondingly the axons were only impregnated in their proximal part, if at all, in the older group. Some of the preparations from adult cats made according to the rapid Golgi technique have nevertheless been fairly successful. The results reached on the material from adult cats have confirmed those obtained from kittens. The distribution and branching of the proximal dendrites were the same regardless of age. The spines were studied mainly with the Golgi-Kopsch technique on kittens up to several months of age. The Golgi-Kopsch preparations have sometimes also shown terminal branching of the afferent axons. The relation between the different LCN neurones was studied in Golgi-Cox preparations from kittens and adult cats. Brain Research, 10 (1968) 352-368
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DISCUSSION
Afferentfibres Most of the afferent fibres to the LCN are rather coarse and enter the nucleus from all sides, although those from the dorsal part of the lateral funiculus dominate. These latter fibres branch richly and have large terminal fields in the nucleus. The contacts are probably made with terminal clusters of boutons and with boutons de passage. Sometimes the afferent fibres are collaterals of vertical fibres and sometimes they are ascending stem axons terminating within the nucleus. There are also a fe~ fine afferent fibres which appear to have smaller terminal fields where boutons de passage are the dominating type of terminal. Whether this means that there are really two different afferent systems is difficult to decide from this material. The fine fibres might be branches of coarse fibres leaving their parent fibre at a rather long distance from the LCN. One observation presented here indicates the existence of coarse descending fibres to the nucleus. One afferent system seems to be the initial collaterals described here. These are very thin, and after branching they terminate within the nucleus with what seems to be small boutons or tree endings. The axons passing dorsally and ventrally to the LCN and which are seen giving off collaterals to the nucleus in transverse sections (Fig. 6) might well be axons from cell bodies located above or below the plane ol" section. These initial collaterals will be further discussed later on. Afferent fibres to the LCN have been described by CajaD in Golgi preparations and also by Ha and Liu 9 1'). Cajal described two types of fibres, coarse and thin. The thin fibres were regarded by him as collaterals of the dorsal spinocerebellar tract terminating with boutons and free endings. Ha and Liu reported from their investigation with a modified Golgi-Cox method three types of terminals within the LCN. boutons de passage, boutons in terminal clusters and synaptic terminals in baskets (basket or calyx of Held). One single fibre could establish contact with more than one neurone, and one neurone could receive numerous terminals of different types and from different fibres. 3-his investigation thus supports the earlier ones in most respects. In my opinion. however, it is not possible to state either from this Golgi material or from that of Cajal 5 or Ha and Liu 9,12 from what fibre tracts the collaterals from the dorsal part of the lateral funiculus emerge. They may just as well be collaterals from a specific spinocervical tract s as from tracts ascending beyond the rostral pole of the LCN. It can be stated, however, that at least some of the afferent fibres are ascending ones terminating within the LCN. N o baskets of Held have been seen with the impregnation methods used here. The afferent fibres have been seen entering the LCN not only frcm the dorsolateral, lateral and ventrolateral side but from all sides. Coarse and thin fibres may have different modes of termination. There may exist coarse fibres descending to the LCN. The final evaluation of these last two findings wilt have to await further neuroanatomical or neurophysiological investigations. Brain Research, 10 (1968) 352-368
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The spinal afferent fibres to the LCN, demonstrated with neuroanatomical degeneration methods 3,31 or with neurophysiological techniquesS,]3,'23,26,3'~, 33 ascend in the dorsal part of the lateral funiculus. They come from all levels of the spinal cord below the nucleus. Electron microscopical investigations (Westman, in preparation) show that most of the boutons within the LCN are unchanged after hemisection immediately below the nucleus and thus indicate the presence of afferent fibres from sources other than below the level of the LCN. One possible explanation for this could be the presence of initial axon collaterals from the LCN neurones. This will be discussed later on. Another explanation is the presence of cutaneous afferents from the trigeminal nerve. Such afferents have been reported in a single neurophysiological study 39. Two types of afferent fibres - - coarse and thin - - have been described here. This is of interest referring to neurophysiological investigations s in which two types of afferent inflow to the LCN are described. LCNneurones
Cajal 5 stated that the LCN neurones have a characteristic appearance in that they are mostly spherical with curved 'hairy' dendrites which remain within the nucleus or its immediate neighbourhood. Furthermore, he noted that the axons, after some deviations, take a vertical course to ascend within the lateral funiculus on the same side. Ha and Liu 9,19 reported that the dendrites mostly seem to run longitudinally r~ and sometimes entered the grey matter of the dorsal horn 9. They also described a peculiar branched dendritic process that existed beside ordinary spines and which established contact with other dendrites or the parent cell body r~. In the present investigation, Cajal's findings regarding the dendrites of the nerve cells in the LCN have been fully confirmed. The axons, however, seem to go ventromedially to enter the grey matter of the spinal cord if followed far enough. This may seem to be in disagreement with Cajal's findings but is in accordance with other investigations (Boivie and Grant, in preparation) using the Nauta method. The reason for this may be that Cajal did not follow the axons far enough. The initial part of the axon is sometimes ascending. In order to follow the axon into the grey matter it is necessary to have longitudinal sections cut obliquely in relation to the median plane. Ordinary spines project from all dendrites, especially from the small ones. This has been found in preparations treated according to the Golgi-Kopsch method, which is considered to be the best one for demonstrating spines. Small projections, without peripheral swellings, can occasionally be seen to emerge from the cell body. in ultrastructural investigations of the L C N 40,41 spines can be demonstrated on dendrites. Other processes similar to those described here seem to emerge from the cell body. Branched spines from the LCN dendrites or the cell bodies have been demonstrated neither with the three modifications of the Golgi method used here, including some perfused specimens, nor with the electron microscope in serial sections 40. One Brain Research, 10 (1968) 352-368
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can therefore not exclude that the micrographs published by Ha and Liu te show axon terminals on LCN neurones. No differences between cats of different ages regarding the dendritic tree have been observed in this study, although such differences have been reported from studies on the cerebral cortex by the Scheibels 34. This development of neurones in young animals might be a phenomenon characteristic of higher brain centres. A description of the LCN neurones based on frozen sections prepared according to the Glees method by Brodal and Rexed a has shown that the cell processes can be 6-8 on each cell. The processes tend to take a longitudinal course within the LCN and are confined to the nucleus. Ha and l_iuv-' also observed the predominantly longitudinal direction of the LCN dendrites and reported in another study 9 that the dendrites could enter the grey matter of the dorsal horn. The present study confirms earlier ones concerning the direction of the LCN dendrites and shows, in accordance with Ha and Liu 9, that the LCN dendrites occasionally enter the grey matter of the neighbouring dorsal horn. The number of cell processes observed in well-impregnated specimens seems to be the same in this investigation as in the one by Brodal and Rexed 3. Earlier Golgi investigations have failed to demonstrate axon collaterals from the nerve cells in the LCN. The reason for this might be their extreme thinness or the fact that immersion fixation and not perfusion was used by earlier investigators of this region. The orientation of the block at the sectioning is another factor which might have affected the earlier negative findings. Some of the axon collaterals seem to end with terminal boutons. 3"he collaterals of motor cell axons in the ventral horn of near-term cat foetuses, on the other hand, end without terminal boutons as demonstrated by Prestige e7. The presence of initial axon collaterals in the LCN fits in well with what is known about the functional organization of the nucleus. Neurophysiologicat studies have shown recurrent inhibition in the LCN alter antidromic s,l~ as well as orthodromic stimulation 13. These findings indicate the presence of internuncial neurones within the nucleus, activated by boutons from recurrent initial collaterals. This would mean that at least three groups of boutons may be present in the nucleus, one from the fibres ascending in the dorsal part of the lateral funiculus, another from the recurrent axon collaterals, and a third from the supposed internuncial neurones. Whether the terminals from the recurrent collaterals and from the internuncial neurones represent all the unchanged boutons in the electron microscopical material 41 after lesions immediately below the LCN, cannot be decided yet. Experimental neuroanatommal investigations (Westman, in preparation) may show whether the LCN receives descending fibres from other levels of the central nervous system. Some of the cells in the medial upper part of the LCN are atypical and resemble the nerve cells in the neighbouring lateral portion of Rexed's lamina V of the dorsal horn. Here the LCN is often connected with the dorsal horn by small strands of cells. as observed already by Rexed a°. While this investigation was being made, perfusion methods for Golgi-Kopsch ~z and for the rapid Golgi method 19 were published. These methods were adopted for Brain Research.
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the last p r e p a r a t i o n s in this study. In addition, two o f the kittens in the G o l g i - C o x material were perfused with the same solution that was later used for i m p r e g n a t i o n . A difference between the immersed and the perfused material was observed only with the r a p i d G o l g i specimens. Thus axon collaterals were d e m o n s t r a t e d exclusively in perfused specimens, although only a small p a r t o f the p r e p a r a t i o n s (5 out o f 68) were perfused. W h e t h e r this means that perfusion is definitely superior to immersion fixation is not possible to decide, especially when the h a z a r d o u s outcome o f the Golgi i m p r e g n a t i o n is considered.
SUMMARY
The afferent fibres to the lateral cervical nucleus ( L C N ) and its nerve cells were studied in Golgi p r e p a r a t i o n s from 94 cats ranging in age from n e w b o r n to adult. The rapid Golgi m e t h o d , the G o l g i - K o p s c h technique and the G o l g i - C o x m e t h o d were used. Some o f the spinal cords were fixed by perfusion. M o s t o f the afferents to the L C N were observed to come from the dorsal p a r t of the lateral funiculus. Some o f them u e r e ascending fibres t e r m i n a t i n g in the nucleus, others were collaterals o f longitudinally oriented fibres, q-he dendrites and their spines are described and also the axons. The latter were found to give off initial collaterals which t e r m i n a t e within the nucleus. Some o f the cells in the medial u p p e r part o f the L C N were atypical and resembled the cells o f the n e i g h b o u r i n g lateral p o r t i o n o f lamina V o f Rexed o f the spinal cord, rather than the other L C N neurones. The findings are discussed in relation to the n o r m a l ultrastructure of the LCN, to some experimental n e u r o a n a t o m i c a l light- and electron microscope data, and to some physiological findings.
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