The collateral projections of spinocerebellar neurons located in the L 2to Ca 1 spinal segments in the cat were investigated by retrograde fluorescent double labeling technique. Rhodamine labeled latex microspheres (Rm) amd Fast Blue (BF) were used for injections into the cerebellum in 8 cats. Two additonal cats, with injections of Fluoro-Gold (FG) combined with Rm were excluded because lipofuchsin autofluorescence obscured the labeling. Aafter injections with one tracer unilaterally in the paramedian lobule and another tracer bilaterally in the anterior lobe, double labeled neurons were found on the side of the paramedian lobule injection in the anterior lobe, double labeled neurons were fond on the side of the paramedian lobule injection in the column of Clarke at L2-L4, laminae IV-VI at L2-L5 and the dorsolateral nucleus at L2-L6. After bilateral injections of one tracer in lobule VIII Band another in the anterior lobe, double labeled neurons were found bilaterally in the column of Clarke at L2-L4, laminas IV-VI at L2-L5, the medial part of lamina VII at L6-L7 and in certain cell groups at sacro-coccygeal levels. Neurons in the lateral nucleus of L4-L5 were labeled exclusively from. injections in the anterior lobe. The findings indicate that spinocerebellar neurons at lumbar and more caudal levels of the cat spinal cod have different projection patterns in the cerebellum. A certain number of neurons which project to the anteriro lobe have divergent axon collaterals supplying also the postrior vermis and/or the paramedian lobule. Other peurons project to the anterior or to the posterior lobe only.

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133+ cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133-cells, these CD133+cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.

The fiber couse of the spinocerebellar tracts in the ventral and lateral funiculi of the cat spinla cord were studied by a new approach making cordotomies at different spinal levels or lesions of the restiform body followed by injectionas of HRP or WGA-HRP into the anterior cerbellar olbe. The retrogradely labeled axons showed characteristic distribution patterns ralated to the level and extent of the lesions. The results show the following. 1) The dorsal spinocerbellar tract (DSCT) originationg ipsilaterally form the thoracic and upper lumbar segments ascends in the dorsolateral fasciculus. It underoes a dorsal shift during its rostral coures. The tract is topially arranged and passes through the restiform body. 2) The ventral spinocerebellar tract (VSCT) arising contralaterally from lower thoractic, lumbar, and more eaudal segments passes via the ventral funiculus and ascends in the ventrolateral fasciculus. This tract is also topically arranged. It maks a lateral and then a dorsal region enetrs via the restiform body. 3) The spnocerebellar fibers oriinationg ipsilaterally from the cervical enlafrgement ascend in the lateralmost part of the lateral funiculus in the area between the dorsolateral and ventrolateral fasciculi, There fibers form two groups, one passing through the dorsolateral and ventrolateral fasciculi. These form two groups, one passing through the restiform body, the other through the superior cerbellar peduncle. 4) The spinocerebellar fibers origination comtralaterally form the central cervical nucaleus pass through the ventral funiculus and ascend in the lateralmost part of the lateral funiculus, mainly inthe ventrolateral fasciculus. Most of the fibers seem to pass through the superior cerebellar peduncle.

Objective To induce significant improvement of motor abnormalitie s and striatal dopamine (DA) levels in rat model of Parkinson's disea se (PD), by intrac2 erebral grafting of the genetically modified muscle cells expressing tyro sine hydroxyla se (TH). Me t h o ds Primary myobla st s and myotube s from the rat were prepared by cell culture and a plasmid, pCMVTH, containing TH gene and a promoter of cytomegalovirus (CMV) wa s constructed by DNA recombination technique. The primary muscle cells were transfected with newly constructed pCMVTH DNA vector, by using lipofection. The se genetically modified muscle cells were grafted into the caudate-putamen of 62OHDA2le sionedrats, repre senting PD models. Before and after grafting, the rotational behaviour and the striatal levels of DA and it s metabolitie s were te sted at different po stoperative survival time s. In addition, the immunocy to chemistry for showing TH activity wa s done both in vitro and in vivo. Results The newly constrcuted pla smid, pCMVTH wa s proved to contain TH gene and have correct direction of insertion. The cultured primary myobla st s and myotube s lipofected with pCMVTH were immunocytochemically shown to expre ss TH activity in vitro. After grafting, the se TH2expre ssing muscle cells showed to have a long2term survival cells in vivo and induced a marked decrea se in abnormal locomotion and a increa se in striatal DA levels for PD rat model. Conclusions In experimental gene therapy for PD, the pCMVTH is a useful vector for carrying TH gene. The lipofection is a practical technique for transferring a target gene into eukaryote s and primary cultured muscle cells should be a good vehicle for DNA transfer and intracerebral grafting.