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Exp Brain Res (2005) 162: 250–256 DOI 10.1007/s00221-004-2132-6 RESEARCH ARTICLE Qunyuan Xu . Gunnar Grant Course of spinocerebellar axons in the ventral and lateral funiculi of the spinal cord with projections to the posterior cerebellar termination area: an experimental anatomical study in the cat, using a retrograde tracing technique Received: 7 May 2004 / Accepted: 28 September 2004 / Published online: 15 December 2004 # Springer-Verlag 2004 Abstract The course of retrogradely labeled spinocerebellar fibers in the ventral and lateral funiculi of the spinal cord was studied following injections of wheat germ agglutinin-conjugated horseradish peroxidase into the posterior spinocerebellar termination area in the cat. Fibers labeled from unilateral injections into the paramedian lobule were found on the same side in the dorsal part of the lateral funiculus (DLF), corresponding to the dorsal spinocerebellar tract (DSCT), but contralaterally in the ventral part of the lateral funiculus (VLF) and in the ventral funiculus (VF), corresponding to the ventral spinocerebellar tract (VSCT). Following injections into the posterior vermis, labeled fibers were less numerous. Most of them were found in the DSCT and only very few in the VSCT. Previously identified cells of origin of these spinocerebellar tracts were labeled in these experiments and counted. They correlated well with the extents and the locations of the injections that had been made into the two termination sites. These results represent novel detailed information on the location of axons projecting to the two main posterior spinocerebellar termination sites in the spinal white matter in the cat. Keywords Ascending pathways . Cerebellum . WGAHRP . Retrograde transport Abbreviations CCN: central cervical nucleus . DL: dorsolateral nucleus . DLF: dorsal part of lateral Q. Xu . G. Grant (*) Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden e-mail: [email protected] Fax: +46-8-325325 Q. Xu Capital Institute of Medicine, Beijing, People’s Republic of China Present address: Q. Xu The Beijing Center for Neural Regeneration and Repairing, Capital University of Medical Sciences, 100054 Beijing, People’s Republic of China funiculus . DSCT: dorsal spinocerebellar tract . RSCT: rostral spinocerebellar tract . VF: ventral funiculus . VL: ventrolateral nucleus . VLF: ventral part of lateral funiculus . VSCT: ventral spinocerebellar tract . VIIB, VIIIA, VIIIB: cerebellar sublobules according to Larsell (1953) Introduction In an earlier study we used a retrograde tracing technique to investigate the location and course of spinocerebellar axons in the spinal cord of the cat following injections of horseradish peroxidase (HRP) or wheat germ agglutinin (WGA)-HRP into the anterior lobe, the main area of termination of spinocerebellar fibers (Grant and Xu 1988a; Xu and Grant 1994). The same technique had also been used in two different studies on the spinocerebellar tracts in the rat (Shirao 1987; Yamada et al. 1991). These studies selectively visualized spinocerebellar axons at spinal level for the first time using an anatomical approach. The same approach, but with the use of Fluoro-Gold instead of HRP or WGA-HRP, was applied later in a study in the North American Opossum, Didelphis virginiana (Terman et al. 1998). Earlier anatomical studies on the location and course of these tracts had been based on anterograde tracing following either spinal cord lesions or injections of tracer into areas of spinocerebellar cell groups. Neither of these had allowed the selective visualization of spinocerebellar fibers. Combining the retrograde tracing with spinal cord lesions at different levels or lesions of the cerebellar peduncles allowed us to define the location and course of axons from different segmental levels. In the present study we have investigated the location and course of spinocerebellar axons projecting to the posterior area of termination, including the paramedian lobule and the adjoining medial part of the dorsal paraflocculus, sublobule VIIIB and parts of sublobules VIIB and VIIIA of the posterior vermis (Grant 1962; Matsushita 1988; Matsushita and Tanami 1987; Matsushita and Yaginuma 1989; Matsushita et al. 1985; Yaginuma and Matsushita 251 1987, 1989; Voogd 1964; Wiksten 1979b, 1987; Xu and Grant 1990). Materials and methods Six adult cats, 3.9–6.5 kg at operation, were used in the present study. The “principles of laboratory animal care” were followed. All surgical procedures were made with the animals under deep anesthesia. This was induced by Rompun (10 mg/kg s.c.; Bayer) followed by Mebumal (30 mg/kg i.p.; Abbott). The animals were fixed in a stereotaxic frame during the operations, which were carried out under sterile precautions. The posterior part of the occipital bone was removed to give access to the needle for injection of the tracer into the cerebellum. The injections were made into the paramedian lobule and/or the posterior vermis, using a Hamilton syringe. WGAHRP (0.8–1.9 μl) was injected and the wound was closed. Following postoperative survival periods of 3–4 days, the animals were reanesthetized deeply and perfused through the ascending aorta with warm (about 36 °C) Tyrode’s solution followed by a fixative containing 1% paraformaldehyde, 1.5% glutaraldehyde and 5% sucrose in 320 mOsm phosphate buffer (pH 7.4) at 4 °C. This was followed by perfusion with 10% sucrose in the same Fig. 1 Diagrams of unfolded cerebellar cortex modified from Grant (1962) showing the injection sites in each case. Solid areas indicate strong WGAHRP activity, stippled areas weak activity (Crus I and Crus II anterior and posterior parts of the ansiform lobule, N.f. fastigial nucleus, N.i. interposed nucleus, p.ant. pars anterior of the paramedian lobule, p.cop. pars copularis, p.post. pars posterior, pfl medial part of dorsal paraflocculus, pmd paramedian lobule, V–IX cerebellar lobules according to Larsell 1953) buffer. The central nervous system was dissected out, and blocks were prepared from the cerebellum, brainstem and cervical (C1, 2, 3, 5, 7, 8), thoracic (T5, 10, 13), lumbar (L2, 3, 5, 6) and sacral (S2) spinal cord segments. The blocks were kept in phosphate buffer containing 30% sucrose (4 °C) overnight and sectioned in series at 60 μm on a freezing microtome. The spinal cord and brainstem sections were cut transversely and collected in groups of ten and five, respectively. Two sections from each group were incubated with tetramethylbenzidine (TMB) according to Mesulam (1978). The cerebellar sections were cut in the sagittal plane and divided into groups of five. One section from each group was incubated with TMB. Labeled axons and neuronal cell bodies in the spinal cord sections were identified in the microscope at ×25 objective magnification in brightfield illumination and mapped, using an X-Y recorder device connected to the microscope, as in our previous study (Xu and Grant 1994). Only cells that were either labeled homogeneously or showed a relatively clear nuclear outline were selected for mapping. All of these cells were entered on summarizing diagrams from the respective segments and counted (see Grant and Xu 1988b). The injection sites, as seen in the cerebellar sections, were examined under the microscope, mostly at ×10 objective magnification, and mapped, using an enlarging projector (×10). The extents of the injections 252 were summarized in diagrams of the unfolded cerebellar cortex (Fig. 1). Results The injections into the cerebellum covered the posterior spinocerebellar termination area to a different extent in different cases (see Fig. 1). Two of them (C255 and C258) comprised the main portion of the paramedian lobule area of termination (the posterior part of this lobule, pars copularis) completely. In one of these (C255), the termination area in the medial part of dorsal paraflocculus was also covered. There was no involvement of the area of termination in the posterior vermis in any of these cases. In another two cases (C254 and C257) the injections comprised the termination site in sublobule VIIIB on both sides, although less extensively in the rostral part in one of them (C254), and not completely on the right side in either of them. The termination site in sublobules VIIB and VIIIA was covered by the injection in one of them (C257). In this case there was also a very minor involvement of the paramedian lobule bilaterally. Another two cases (C253 and C256) had injections covering sublobules VIIB, VIIIA and VIIIB, and parts of the termination area in the paramedian lobule, most prominently in one of them (C253). In this case, the medial part of dorsal paraflocculus was involved too. In both of these cases there was also a slight involvement of a lateroposterior part of the fastigial nucleus and of the posterior interposed nucleus. Two of the cases (C256 and 257) showed some spread of tracer to the dorsal surface of the brainstem on the left side, including the dorsal part of the most caudal portion of the inferior vestibular nucleus. Labeled axons were found in the lateral and ventral funiculi of the spinal white matter in all six cases. The findings in case C255, in which the injections had affected both the paramedian lobule area of termination and that of dorsal paraflocculus, will be described in detail first. Labeled axons were present bilaterally in the spinal white matter (Fig. 2). On the right side, the side of the injection, they were found in the dorsal part of the lateral funiculus (DLF), and on the left side, in the ventral part of the lateral funiculus (VLF) and in the ventral funiculus (VF). The amount of labeled fibers in the DLF was larger than in the VLF. The findings in case C258 were almost identical. In the DLF, labeled axons were present from above the L3 segment (Fig. 2). They were located peripherally, with fibers adding from deeper parts at thoracic and up to mid-cervical levels. On the left side, labeled axons were found in the VF at lower lumbar levels (L3–L6), but more rostrally, up to upper cervical segments, in the peripheral parts of the VLF. A large number of labeled neuronal cell bodies (116) were found in the column of Clarke and in laminae IV–VI on the right side (T5–L3), and some (19) on that side in laminae V–VI at the level of the cervical enlargement (C5–C8). On the same side, there were many (41) labeled neuronal cell bodies in the dorsolateral nucleus (DL; Grant et al. 1982) Fig. 2 Diagrams showing distributions of WGA-HRP labeled axons and perikarya for case C255. Each drawing represents one section. Notches are on the right side dorsally at L3–L6, a few (six on the left, two on the right) in the medial group of lamina VII neurons at lumbosacral levels (L5–S2; Grant et al. 1982) and some (16 on the left, six on the right) in the central cervical nucleus (CCN; C1–C3). In two of the cases with injections affecting the termination area in the posterior vermis (C254 and 257), there were only small amounts of labeled axons, particularly in the one in which sublobule VIIIB was less affected (C254). In this case, the termination sites in sublobules VIIB and VIIIA were not involved at all. In the case with less sparse labeling (C257; not illustrated), most of the labeled axons were found in the DLF, mainly on the left side, only very few bilaterally in the VLF, and only single ones in the VF on the two sides. The single ones in the VF were found at lower lumbar and at upper cervical levels. Labeled neuronal cell bodies were present in small amounts bilaterally in the medial group of lamina VII at lumbosacral levels (17 on the left, 10 on the right; L5–S2). Larger amounts were found in Clarke’s column and laminae IV–VI at thoracolumbar levels (T5–L3), predominantly on the left side (40 versus 10). Moreover, there were a large number of labeled neurons in the CCN. Most of these were on the right side (118 versus 79; C1–C3). Of the two remaining cases, C253 and C256— representing sublobule VIIIB plus paramedian lobule (C253) injections—case C253, which had the most extensive affection of the posterior spinocerebellar termination sites, will be described in detail. In this case, labeled axons were present bilaterally, both in the DLF and in the VLF and VF (Fig. 3). There was an asymmetry, however, in the sense that more axons were found in the VLF and VF on the right side but in the DLF on the left. The latter was the side where the injection had covered the termination site in the paramedian lobule most extensively. 253 Discussion Retrogradely labeled fiber tracts Fig. 3 Diagrams showing distributions of WGA-HRP labeled axons and perikarya for case C253. Each drawing represents one section. Notches are on the right side dorsally Otherwise, with one exception, the locations of the labeled fibers were similar to what was found in case C255. The exception concerned fibers appearing bilaterally in the VF at upper cervical levels, which were not seen in case C255. There was a large number of labeled neuronal perikarya in the CCN in case C253 (153 on the left side, and 146 on the right; C1–C3), but just a few (16 and 6, respectively) in C255. Furthermore, there was a large number (245 on the left side, 218 on the right) in Clarke’s column and laminae IV–V at thoracic and upper lumbar levels (T5–L3), which means twice as many perikarya with these loctions as in case C255. About twice as many labeled neurons as in case C255 were also found in the DL nucleus at L3–L6 (86 on the left, 31 on the right). Another striking difference when compared with case C255 was that the medial group of lamina VII had rather many labeled cell bodies in case C253 (64 on the left, 65 on the right; L5– S2), but just a few (six and two, respectively) in C255. Case C256 had labeled axons distributed similarly to case C253, although there was no clear asymmetry with regard to the amount of labeled fibers in the VLF and VF and in the DLF on the two sides. More axons were found in the VF, however, particularly in the upper cervical segments, but also at lower lumbar levels. There were many more labeled neurons in the CCN (232 on the left, 255 on the right), but fewer medially in lamina VII at lumbo-sacral levels (29 on the left, 30 on the right) and in the DL nucleus at L3–L6 (47 on the left, 22 on the right). The findings in the present study further demonstrate the usefulness of selective retrograde labeling of spinal fiber tracts following injections into areas of termination. In a previous study in the cat we localized spinocerebellar fibers with projections to the anterior lobe of the cerebellum, the main area of termination of the spinocerebellar tracts (Xu and Grant 1994). This was also done later in the North American Opossum, Didelphis virginiana (Terman et al. 1998). Here we have examined the locations of spinocerebellar fibers projecting to the less extensive, posterior termination area. This includes two main sites, the posterior part (pars copularis) of the paramedian lobule, with the adjoining medial portion of dorsal paraflocculus, and parts of posterior vermis, including sublobule VIIIB and portions of sublobules VIIB and VIIIA (Grant 1962; Voogd 1964; Matsushita and Tanami 1987; Matsushita and Yaginuma 1989; Matsushita et al. 1985; Yaginuma and Matsushita 1987, 1989; Wiksten 1979b, 1987). Retrogradely labeled axons were found from both of these sites of termination, although only in small amounts following injections into the posterior vermis. The locations of the labeled fibers corresponded to the sites of the classical dorsal and ventral spinocerebellar tracts (DSCT and VSCT, respectively; see for example Xu and Grant 1994). The DSCT is found peripherally in the DLF, with fibers adding from deeper parts at more rostral levels, where additional cells of origin are found. The VSCT is located in the VF at sacral and lower lumbar levels and peripherally in the VLF at more rostral levels. At the level of the cervical enlargement there were contributions of fibers to the mid peripheral area of the lateral funiculus, corresponding to the location of the rostral spinocerebellar tract (RSCT; Matsushita et al. 1985; Wiksten 1985; Wiksten and Grant 1986; Xu and Grant 1994). Finally, at high cervical levels labeled fibers appeared in the VF in some of the cases. These could be related to the CCN, which is present at C1–C4 in the cat (Rexed 1954). It gives rise to crossing spinocerebellar axons (Matsushita et al. 1979; Matsushita and Ikeda 1980; Wiksten 1979a, 1987; Xu and Grant 1994). Retrogradely labeled neuronal cell groups Counting of labeled neuronal perikarya in the spinocerebellar cell groups was carried out in the different cases in the same way as in our previous study (Xu and Grant 1994). This created a useful support for interpreting variations in relative amounts of labeled axons at different levels and locations. These cell groups have been identified anatomically mainly by retrograde tracing methods in the cat (Grant et al. 1982; Grant and Xu 1988a, 1988b; Matsushita et al. 1979; Matsushita and Ikeda 1980; Wiksten 1975, 1979b, 1985; Xu and Grant 254 1994). Cells in the column of Clarke and laminae IV–VI from L3 to upper thoracic levels give rise to uncrossed fibers to the DSCT. The medial group of lamina VII at lumbosacral levels and the DL nucleus at L3–L6 are sources of the VSCT, the former with both crossed and uncrossed axons, the latter with crossed axons. The VSCT also originates from cells in the ventrolateral nucleus (VL) in L4–L5, together with cells in a lateral group of lamina VII, close to the DL nucleus (Grant et al. 1982). These groups, however, which were not labeled in the present experiments, project exclusively to the anterior lobe and not to the posterior spinocerebellar termination sites (Xu and Grant 1988). The RSCT takes its origin ipsilaterally from cells in laminae V–VI at C5–C8. At upper cervical levels, the CCN is a source of crossing spinocerebellar axons. Spinocerebellar fibers labeled from the area of the paramedian lobule (C255 and C258) The paramedian lobule injections (C255 and C258) resulted in labeled fibers distributed bilaterally, but with different locations. They were found in the DLF on the side of the injection and in the VF and VLF on the other side. The amount of fibers seemed to be larger in the DLF than in the VLF. This is in agreement with the previouslyreported finding that the DSCT contributes more heavily to the projection to the paramedian lobule than the VSCT (Grant 1962). The ipsilateral fiber labeling could be correlated to labeled cell bodies distributed ipsilaterally in the column of Clarke and laminae IV-VI at thoracolumbar levels and ipsilateral cells originating from the RSCT at the level of the cervical enlargement. This would confirm earlier findings by Matsushita and Ikeda (1980), who found ipsilateral labeling of cells with these locations following HRP injections into the paramedian lobule. The contralateral fibers, in the VLF and VF, which appeared from levels located far caudally, could be correlated to cells originating from the DL nucleus on the side of the injection, with axons crossing to the contralateral side of the spinal cord, and to cells located bilaterally in the medial group of lamina VII neurons at lumbosacral levels. Cells from the DL nucleus projecting to the paramedian lobule have been identified before (Xu and Grant 1988). They correspond to the retrogradely labeled spinal border cells and lateral lumbar nucleus neurons that were found in the study by Matsushita and Ikeda (1980), following HRP injections into the paramedian lobule. They found that the axons cross twice, first at spinal level and then inside the cerebellum, and therefore concluded that the projection to the paramedian lobule was ipsilateral to the cells of origin. Like Matsushita and Ikeda (1980), we have previously demonstrated that axons from the DL nucleus (spinal border cells and lateral lumbar nucleus of Matsushita and Ikeda) project contralaterally in the spinal cord (Grant et al. 1982; Grant and Xu 1988b). Furthermore, it is well established that the VSCT has a mainly contralateral termination in the cerebellum (Grant 1962; Voogd 1964). Our results are therefore in complete agreement with the conclusion of Matsushita and Ikeda. Some neurons were also labeled bilaterally in the CCN, although their axons were not identified in the ventral white matter. A bilateral labeling of CCN neurons is in agreement with the observations reported by Matsushita and Ikeda (1980). They found occasional neurons labeled in the CCN on both sides following injections into sublobules A2 and B1 (corresponding to parts of pars anterior and pars posterior) of the paramedian lobule. Furthermore, anterograde labeling with WGA-HRP following spinal cord hemisections above the level of injections into the CCN has demonstrated bilateral projections to the paramedian lobule, although predominantly contralateral to the cells of origin (Matsushita and Tanami 1987). Spinocerebellar fibers labeled from posterior vermis (C254 and C257) The posterior vermis injections (C254 and C257) gave rise to only small amounts of labeled fibers, particularly in the case in which sublobule VIIIB was less affected and the termination sites in sublobules VIIB and VIIIA were not involved (C254). In the case with less sparse labeling, most of the labeled fibers were located in the DLF, and only very few in the VLF and VF. This is in agreement with the previously-reported finding that the DSCT contributes more heavily to the projection to the posterior vermis than the VSCT (Grant 1962). The small amount of labeled neurons in the medial group of lamina VII at lumbosacral levels goes in parallel with the very few labeled axons in the VLF, and also in the VF at lower lumbar levels. Matsushita and Ikeda (1980) found this group to project to sublobule VIIIB. In the DLF, there were more labeled fibers on the left side. This is reasonably explained by the more extensive covering by the injection of the vermis on that side. There were also more labeled neuronal cell bodies in the column of Clarke and laminae IV–VI on that side. A projection from the column of Clarke to sublobule VIIIB was described earlier (Matsushita and Ikeda 1980). The large number of labeled neurons in the CCN explains the occurrence of labeled axons in the VF at upper cervical levels. That these neurons were mainly on the right side would be expected, considering the contralateral dominance of the vermal injection. A CCN projection to the posterior vermis was described earlier (Wiksten 1979b, 1987; Matsushita and Ikeda 1980). Spinocerebellar fibers labeled from injections affecting both the posterior vermis and the paramedian lobule (C253 and C256) The injections which affected both the paramedian lobule and the posterior vermis (C253 and C256) resulted in labeled fibers located bilaterally in both the DLF and the VLF and VF. In the case with the most extensive injection 255 (C253) there was a clear asymmetry. More labeled axons were found in the DLF on the left side but in the VLF and VF on the right. This should reasonably be explained by the more extensive involvement of the paramedian lobule on the left side. In analogy with the findings in case C255, this termination site should receive afferents from the DSCT on the left side and from the VSCT on the right. In contrast to the finding in case C255, there were labeled axons distributed bilaterally in the VF at high cervical levels. This is reasonably explained by the presence of a large number of labeled neurons located bilaterally in the CCN, as opposed to just a few in case C255. That the labeled axons would be descending fibers, originating in the left fastigial nucleus, which was involved by the injection, should be possible to exclude. Labeled axons were found bilaterally and the fastigiospinal projection has been found to be exclusively unilateral, contralateral (Fukushima et al. 1977; Matsushita and Hosoya 1978). That there was a large number of labeled neurons in the CCN in case C253 might be related to the projection to the posterior vermis. The injection into this part of the cerebellum in case C257 resulted in a large number of labeled neurons in the CCN. On the contrary, an injection into the paramedian lobule, in case C255, gave rise to just a few. The large number of labeled neurons in Clarke’s column and laminae IV–VI, compared to the situation in case C255, might be explained by the involvement of both the paramedian lobule and the posterior vermis, each of which receive projections from these groups of neurons, as discussed above. A retrograde labeling of some neurons in these groups derived from collaterals of spinocerebellar axons projecting to the fastigial and interposed nuclei cannot be ruled out, however, considering the finding by Matsushita and Ikeda (1970) that the DSCT sends collaterals to these nuclei. They reported, furthermore, that a similar projection was found also for the VSCT, and that this tract represented the most important connection of the two tracts. This could account for the larger number of labeled neurons in the DL nucleus, compared to case C255. The distribution of labeled axons in case C256 was similar to that in case C253, except that there were more axons in the VF, particularly at upper cervical levels. This could be explained by the larger number of CCN neurons at these levels. No clear explanation can be offered, however, for the larger amount of labeled axons in the VF at lower lumbar levels. The lower number of labeled cells in the medial group of lamina VII neurons, as well as in the DL group, is most probably explained by the less extensive involvement of the paramedian lobule compared to case C253. Comparisons with findings of retrograde labeling from the anterior lobe An interesting finding in the present study was the asymmetry with regard to locations of fibers in the DLF versus the VLF and VF, following injections into the paramedian lobule. This may be expected, however, considering the fact that fibers in the VSCT will have to cross twice, first at their level of origin in the spinal cord, and then again inside the cerebellum, whereas those of the DSCT should pass uncrossed. Unilateral injections of tracer into the anterior lobe, which also receives projections from both the DSCT and the VSCT, should result in the same pattern. In our previous study on retrogradely labeled fibers following injections into the anterior lobe, such injections were however not made (Xu and Grant 1994). With regard to retrogradely labeled cell bodies in different spinocerebellar cell groups, many such cells were found in the present study in the same groups as in our previous study. This may be expected, considering the results of another study, which dealt with collateral projections from the lower part of the spinal cord to anterior and posterior cerebellar termination areas (Xu and Grant 1988). One principal difference, however, was that no labeled neurons were found in the VL nucleus at L4– L5, nor in the lateral group of lamina VII, close to the DL nucleus, as discussed above. Those neurons project exclusively to the anterior lobe and not to the posterior spinocerebellar termination area (Xu and Grant 1988). Conclusions The findings in this study give novel detailed information on the location of axons projecting to the two main posterior spinocerebellar termination sites in the spinal white matter in the cat. Fibers labeled from unilateral injections into the paramedian lobule were found on the same side in the dorsal part of the lateral funiculus (DLF), corresponding to the dorsal spinocerebellar tract (DSCT), but contralaterally in the ventral part of the lateral funiculus (VLF) and in the ventral funiculus (VF), corresponding to the ventral spinocerebellar tract (VSCT). Following injections into the posterior vermis, labeled fibers were less numerous. Most of them were found in the DSCT and only very few in the VSCT. Previously identified cells of origin of these spinocerebellar tracts were labeled in these experiments and counted. They correlated well to the extents and locations of the injections that had been made into the two termination sites. 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