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Spino-olivary neurones in the lumbo-sacral cord of the cat demonstrated by retrograde transport of horseradish peroxidase
176
Brain Research, 168 (1979) 176-179 :C Elsevier/North-Holland Biomedical
Spino-olivary
neurones in the lumbo-sacral
retrograde transport of horseradish
cord of the cat demonstrated
Press
by
peroxidase
D. M. ARMSTRONG and R. F. SCHILD Department
of Physiology,
The Medical
School,
University
of Bristol,
Bristol BS8 1 TD. ( II. K.i
(Accepted January 18th, 1979)
Several spino-olivo-cerebellar pathways (VF-SOCPs) are mediated via spinal axons which ascend to the inferior olive in the ventral funiculus and terminate in the dorsal and medial accessory olives (DA0 and MAO) (e.g. ref. 2). Degeneration experiments3 indicate that most of these axons cross soon after their origin, but the location of the cell bodies within the spinal grey is unknown. Several spatially separate populations of spino-olivary cells may exist however because Oscarsson and Sjalund697 have identified as many as 5 VF-SOCPs with distinctly different functional characteristics. Four paths, denoted a, bs, cl and cs, arise at the lumbo-sacral level whilst only one forelimb path (bl) is known. We report an attempt to localize the lumbo-sacral spino-olivary cells in the cat by the method of retrograde axonal transport of horseradish peroxidase (HRP). In cats anaesthetized with sodium pentobarbitone, injections of HRP were made into one inferior olive (9 cases) or into adjacent regions for control purposes (3 cases). In the olivary experiments and one control case the injections were made through the ventral surface of the medulla; the remaining control injections were made from the dorsal surface of the brain stem. In each case a single injection of from 0.1 to 0.4 ~1 of a 3&50x aqueous solution of HRP (Boehringer; Grade 1) was made using a 1 ~1 syringe. After 3-4 days survival, the cat was perfuse-fixed with 4% glutaraldehyde in phosphate buffer, 30 ,um frozen sections were cut transversely and every tenth section through the injection site and every twentieth from cord segments Ll to S1 inclusive was processed to demonstrate HRP4. After an olivary injection the neuropil in some part of the nucleus was always heavily loaded with brown reaction product and numerous fibres and olivary neurones were labelled. There was never any passive spread of HRP across the midline, but a few neurones in the contralateral olive were always labelled, presumably as a result of retrograde transport in injured olivary axons decussating through the injection site. After all olivary injections retrogradely labelled cell bodies were present in the lumbo-sacral cord, and of 640 found 614 were contralateral to the injection (96 %). The density of reaction product varied widely between cells: many were densely labelled but others contained only a few pale granules. The number of labelled
177 neurones was positively correlated with the size of the injection site and after the 3 largest injections 176, 142 and 86 labelled cells were found. The cell distributions in 3 segments from case 16 with a large injection are shown in Fig. lA, B and C (injection site shown in Fig. 1G), whilst the results for the same segments in all 9 cases are pooled in Fig. lD, E and F. Taken in sum our injections involved all those olivary regions known to receive spinal afferents. Fig. lD, E and F and the corresponding results for other segments revealed that labelled cells were concentrated in 3 different regions of the grey matter. Cells in the dorsal horn were concentrated in the medial two-thirds of laminae IV and V (see Fig. 1A, B, D and E). They were confined to segments LB- LS inclusive. A second ‘intermediate’ population (Fig. lB, C, D, E and F) was more diffusely distributed, both rostrocaudally (Ll- S1 inclusive) and in the transverse plane (scattered throughout the medial half of lamina VII and the dorsomedial part of lamina VIII). By contrast, a third (ventromedial) population lay close against the medial border of the ventral horn at the junction between laminae VII and VIII. These cells were very tightly clustered so that 6 or 7 contiguous neurones were sometimes labelled on a single section. In 3 cases such cells were confined to segments Ls, Ls and L7, in one case to Ls and Ls, in two to LS and L7 and in the remaining case to L6 alone. Though in the case illustrated and in two others cells were found in all 3 regions, in the other cases the injection site was more restricted and labelled cells were confined to only two of the regions (dorsal and intermediate cells in two cases; intermediate and ventromedial cells in 4 cases). Transport in spino-olivary axons seems the most likely explanation for the observed spinal labelling but two other possibilities must nevertheless be considered. Firstly, some labelling might result from HRP uptake by injured axons which may ascend through the olive without providing terminals. In one case therefore, a control injection was made just rostra1 to the olive but no labelled cells were detected in the cord. Secondly, HRP might spread to spino-reticular axons terminating dorsal to the olivess. In 5 cases the injection site was confined within the olive (and together these injections labelled cells in all 3 populations) but in the remainder there was some spread beyond the olive. In 3 cases this was sufficient to involve the ventral-most portions of the paramedian and ventral reticular nuclei. Large control injections were therefore made in two animals into the brain stem overlying one olive. The ventral limit of the larger injection site is shown by a broken line in Fig. 1G. This injection labelled 103 neurones of which as many as 62 were ipsilateral. The paucity of ipsilateral cells in our previous cases therefore argues strongly that there was little HRP transport by spino-reticular axons after olivary injections. Nevertheless, 41 control-case cells (40 %) were contralateral and 26 were within the 3 areas in which cells were found after olivary injections (8 dorsal, 8 intermediate and 10 ventromedial cells). Clearly, therefore, none of these loci is occupied exclusively by cells projecting only to the olive. The other control injection yielded essentially similar results. Our results provide ground for recognizing 3 distinct concentrations of lumbosacral spino-olivary neurones which differ in location in the cord cross-section, in
G
CASE
16
POOLED
caudal
rostra1 1.2
Fig. 1. A, B and C: distribution of HRP-positive cell bodies in 3 segments of the lumbar cord in case 16. D, E and F: distributions of HRP-positive cells in the same segments, as derived by pooling the 9 cases. Scale applies throughout A-F. G : transverse sections to show injection site (stippling) from case 16. Numerals indicate distances in mm between the sections. Abbreviations: PO, principal olive; MAO, medial accessory olive; DAO, dorsal accessory olive; VLO, ventrolateral outgrowth; P, medullary pyramid; LRN, lateral reticular nucleus; XII, hypoglossal axons; vertical line, midline; for broken line see text.
179 rostrocaudal extent and also in packing density. However, since none of our injections was restricted to a single olivary subnucleus we can reach no firm conclusion regarding the possibility that these populations project to different parts of the olive. Only for dorsal horn cells was it useful to compare injections which gave positive and negative results: to label large numbers of these cells it was necessary to involve the rostra1 half of DAO. Despite this difficulty our results invite the speculation that each population constitutes the spinal relay for one of the hindlimb VF-SOCPs defined by Oscarsson and Sjiilunds. If this is the case it seems probable that our dorsal cells relay either the cl or cs path since both synapse in the rostra1 half of DAO. Indeed since these paths partially merge in the olive and have similar (monosynaptic) segmental delays and receptive field characteristics ~7 it is likely that relay cells for both paths have been labelled. As regards the other cells, our results do not show which population might relay which of the bs and a paths. However, only a b path is known for the forelimbs, so that the distribution of spino-olivary cells in the cervical enlargement might, by analogy, identify the lumbo-sacral cells which relay path ba. On this basis our preliminary results from the cervical cord suggest that ‘intermediate’ lumbo-sacral cells may relay path b2. R.F.S. was supported by the Medical Research Council of Great Britain.
1 Armstrong, D. M., Harvey, R. J. and Schild, R. F., Topographical localisation in the olivo-cerebellar projection: an electrophysiological study in the cat, J. camp. Neural., 154 (1974) 287-302. 2 Boesten, A. J. P. and Voogd, J., Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat, J. camp. Neural., 161 (1975) 215-238. 3 Brodal, A., Walberg, F. and Blackstad, T., Termination of spinal afferents to the inferior olive in the cat, J. Neurophysiol., 13 (1950) 431454. 4 Graham, R. C. and Kamovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 5 Maunz, R. A., Pitts, N. G. and Peterson, B. W., Cat spinoreticular neurons: locations, responses and changes in responses during repetitive stimulation, Brain Research, 148 (1978) 365-379. 6 Oscarsson, 0. and Sjiilund, B., The ventral spino-olivocerebellar system in the cat. I. Identification of five paths and their termination in the cerebellar anterior lobe, Exp. Brain Res., 28 (1977) 469-486. 7 Oscarsson,
0. and Sjiilund, B., The ventral spino-olivocerebellar system in the cat. III. Functional characteristics of the five paths, Exp. Brain Res., 28 (1977) 505-520. 8 Rossi, G. F. and Brodal, A., Terminal distribution of spinoreticular fibers in the cat, Arch. Neural. Psychiat. (Chic.), 78 (1957) 439453.
Brain Research, 168 (1979) 176-179 :C Elsevier/North-Holland Biomedical
Spino-olivary
neurones in the lumbo-sacral
retrograde transport of horseradish
cord of the cat demonstrated
Press
by
peroxidase
D. M. ARMSTRONG and R. F. SCHILD Department
of Physiology,
The Medical
School,
University
of Bristol,
Bristol BS8 1 TD. ( II. K.i
(Accepted January 18th, 1979)
Several spino-olivo-cerebellar pathways (VF-SOCPs) are mediated via spinal axons which ascend to the inferior olive in the ventral funiculus and terminate in the dorsal and medial accessory olives (DA0 and MAO) (e.g. ref. 2). Degeneration experiments3 indicate that most of these axons cross soon after their origin, but the location of the cell bodies within the spinal grey is unknown. Several spatially separate populations of spino-olivary cells may exist however because Oscarsson and Sjalund697 have identified as many as 5 VF-SOCPs with distinctly different functional characteristics. Four paths, denoted a, bs, cl and cs, arise at the lumbo-sacral level whilst only one forelimb path (bl) is known. We report an attempt to localize the lumbo-sacral spino-olivary cells in the cat by the method of retrograde axonal transport of horseradish peroxidase (HRP). In cats anaesthetized with sodium pentobarbitone, injections of HRP were made into one inferior olive (9 cases) or into adjacent regions for control purposes (3 cases). In the olivary experiments and one control case the injections were made through the ventral surface of the medulla; the remaining control injections were made from the dorsal surface of the brain stem. In each case a single injection of from 0.1 to 0.4 ~1 of a 3&50x aqueous solution of HRP (Boehringer; Grade 1) was made using a 1 ~1 syringe. After 3-4 days survival, the cat was perfuse-fixed with 4% glutaraldehyde in phosphate buffer, 30 ,um frozen sections were cut transversely and every tenth section through the injection site and every twentieth from cord segments Ll to S1 inclusive was processed to demonstrate HRP4. After an olivary injection the neuropil in some part of the nucleus was always heavily loaded with brown reaction product and numerous fibres and olivary neurones were labelled. There was never any passive spread of HRP across the midline, but a few neurones in the contralateral olive were always labelled, presumably as a result of retrograde transport in injured olivary axons decussating through the injection site. After all olivary injections retrogradely labelled cell bodies were present in the lumbo-sacral cord, and of 640 found 614 were contralateral to the injection (96 %). The density of reaction product varied widely between cells: many were densely labelled but others contained only a few pale granules. The number of labelled
177 neurones was positively correlated with the size of the injection site and after the 3 largest injections 176, 142 and 86 labelled cells were found. The cell distributions in 3 segments from case 16 with a large injection are shown in Fig. lA, B and C (injection site shown in Fig. 1G), whilst the results for the same segments in all 9 cases are pooled in Fig. lD, E and F. Taken in sum our injections involved all those olivary regions known to receive spinal afferents. Fig. lD, E and F and the corresponding results for other segments revealed that labelled cells were concentrated in 3 different regions of the grey matter. Cells in the dorsal horn were concentrated in the medial two-thirds of laminae IV and V (see Fig. 1A, B, D and E). They were confined to segments LB- LS inclusive. A second ‘intermediate’ population (Fig. lB, C, D, E and F) was more diffusely distributed, both rostrocaudally (Ll- S1 inclusive) and in the transverse plane (scattered throughout the medial half of lamina VII and the dorsomedial part of lamina VIII). By contrast, a third (ventromedial) population lay close against the medial border of the ventral horn at the junction between laminae VII and VIII. These cells were very tightly clustered so that 6 or 7 contiguous neurones were sometimes labelled on a single section. In 3 cases such cells were confined to segments Ls, Ls and L7, in one case to Ls and Ls, in two to LS and L7 and in the remaining case to L6 alone. Though in the case illustrated and in two others cells were found in all 3 regions, in the other cases the injection site was more restricted and labelled cells were confined to only two of the regions (dorsal and intermediate cells in two cases; intermediate and ventromedial cells in 4 cases). Transport in spino-olivary axons seems the most likely explanation for the observed spinal labelling but two other possibilities must nevertheless be considered. Firstly, some labelling might result from HRP uptake by injured axons which may ascend through the olive without providing terminals. In one case therefore, a control injection was made just rostra1 to the olive but no labelled cells were detected in the cord. Secondly, HRP might spread to spino-reticular axons terminating dorsal to the olivess. In 5 cases the injection site was confined within the olive (and together these injections labelled cells in all 3 populations) but in the remainder there was some spread beyond the olive. In 3 cases this was sufficient to involve the ventral-most portions of the paramedian and ventral reticular nuclei. Large control injections were therefore made in two animals into the brain stem overlying one olive. The ventral limit of the larger injection site is shown by a broken line in Fig. 1G. This injection labelled 103 neurones of which as many as 62 were ipsilateral. The paucity of ipsilateral cells in our previous cases therefore argues strongly that there was little HRP transport by spino-reticular axons after olivary injections. Nevertheless, 41 control-case cells (40 %) were contralateral and 26 were within the 3 areas in which cells were found after olivary injections (8 dorsal, 8 intermediate and 10 ventromedial cells). Clearly, therefore, none of these loci is occupied exclusively by cells projecting only to the olive. The other control injection yielded essentially similar results. Our results provide ground for recognizing 3 distinct concentrations of lumbosacral spino-olivary neurones which differ in location in the cord cross-section, in
G
CASE
16
POOLED
caudal
rostra1 1.2
Fig. 1. A, B and C: distribution of HRP-positive cell bodies in 3 segments of the lumbar cord in case 16. D, E and F: distributions of HRP-positive cells in the same segments, as derived by pooling the 9 cases. Scale applies throughout A-F. G : transverse sections to show injection site (stippling) from case 16. Numerals indicate distances in mm between the sections. Abbreviations: PO, principal olive; MAO, medial accessory olive; DAO, dorsal accessory olive; VLO, ventrolateral outgrowth; P, medullary pyramid; LRN, lateral reticular nucleus; XII, hypoglossal axons; vertical line, midline; for broken line see text.
179 rostrocaudal extent and also in packing density. However, since none of our injections was restricted to a single olivary subnucleus we can reach no firm conclusion regarding the possibility that these populations project to different parts of the olive. Only for dorsal horn cells was it useful to compare injections which gave positive and negative results: to label large numbers of these cells it was necessary to involve the rostra1 half of DAO. Despite this difficulty our results invite the speculation that each population constitutes the spinal relay for one of the hindlimb VF-SOCPs defined by Oscarsson and Sjiilunds. If this is the case it seems probable that our dorsal cells relay either the cl or cs path since both synapse in the rostra1 half of DAO. Indeed since these paths partially merge in the olive and have similar (monosynaptic) segmental delays and receptive field characteristics ~7 it is likely that relay cells for both paths have been labelled. As regards the other cells, our results do not show which population might relay which of the bs and a paths. However, only a b path is known for the forelimbs, so that the distribution of spino-olivary cells in the cervical enlargement might, by analogy, identify the lumbo-sacral cells which relay path ba. On this basis our preliminary results from the cervical cord suggest that ‘intermediate’ lumbo-sacral cells may relay path b2. R.F.S. was supported by the Medical Research Council of Great Britain.
1 Armstrong, D. M., Harvey, R. J. and Schild, R. F., Topographical localisation in the olivo-cerebellar projection: an electrophysiological study in the cat, J. camp. Neural., 154 (1974) 287-302. 2 Boesten, A. J. P. and Voogd, J., Projections of the dorsal column nuclei and the spinal cord on the inferior olive in the cat, J. camp. Neural., 161 (1975) 215-238. 3 Brodal, A., Walberg, F. and Blackstad, T., Termination of spinal afferents to the inferior olive in the cat, J. Neurophysiol., 13 (1950) 431454. 4 Graham, R. C. and Kamovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 5 Maunz, R. A., Pitts, N. G. and Peterson, B. W., Cat spinoreticular neurons: locations, responses and changes in responses during repetitive stimulation, Brain Research, 148 (1978) 365-379. 6 Oscarsson, 0. and Sjiilund, B., The ventral spino-olivocerebellar system in the cat. I. Identification of five paths and their termination in the cerebellar anterior lobe, Exp. Brain Res., 28 (1977) 469-486. 7 Oscarsson,
0. and Sjiilund, B., The ventral spino-olivocerebellar system in the cat. III. Functional characteristics of the five paths, Exp. Brain Res., 28 (1977) 505-520. 8 Rossi, G. F. and Brodal, A., Terminal distribution of spinoreticular fibers in the cat, Arch. Neural. Psychiat. (Chic.), 78 (1957) 439453.