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Previous studies have shown that intrastriatal transplants of dopamine (DA)-rich fetal ventral mesencephalic (VM) tissue can correct denervation-induced changes in the cellular expression of neuropeptide and receptor mRNAs in the rat Parkinson model. However, with the standard transplantation approach normalization of all cellular parameters has not been obtained. This may be due either to the incomplete striatal reinnervation achieved by these transplants, or to the ectopic placement of the grafts. In the present study we have used a microtransplantation approach to obtain a more complete reinnervation of the denervated striatum (20 micrograft deposits spread over the entire structure). Neurons were also implanted directly into the substantia nigra. In rats with multiple intrastriatal VM transplants the lesion-induced upregulation of mRNAs encoding for preproenkephalin (PPE), the D(2)-type DA-receptor, and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD(67)) was normalized throughout the striatum, whereas the lesion-induced downregulation of preprotachykinin mRNA was unaffected. Intranigral grafts of either fetal DA-rich VM tissue or GABA-rich striatal tissue did not induce any changes in striatal neuropeptide and D(2)-receptor mRNA expression despite significant behavioral improvement. Comparison of the behavioral data with levels of neuropeptide expression showed that in rats with intrastriatal VM transplants a complete normalization of striatal PPE and GAD(67) mRNA expression did not translate into a complete recovery of spontaneous motor behaviors. The results show that extensive DA reinnervation of the host striatum by multiple VM microtransplants is insufficient to obtain full recovery of all lesion-induced changes at both the cellular and the behavioral level. A full reconstruction of the nigrostriatal pathway or, alternatively, modulation of basal ganglia function by grafting in non-striatal regions may be required to further improve the functional outcome in the DA-denervated brain.

Embryonic striatal precursors grafted into the lesioned adult host striatum show limited integration with little migration and restricted efferent projections. In the present study, the influence of an immature striatal environment on the integrative capacity of grafted neuroblasts was examined after transplantation of striatal progenitors into the striatum at different stages of postnatal development. Mouse progenitors, derived from embryonic day 13.5-14 lateral or medial ganglionic eminence or the cerebellar primordium, were transplanted as a single cell suspension into the developing postnatal day 1, 7 and 21 rat striatum. The grafted cells and their axonal projections were visualized using antibodies raised against the mouse-specific neural markers, M6 and M2. Cells from the lateral (but not the medial) ganglionic eminence showed a remarkable capacity to innervate selectively the striatal target structures, globus pallidus, entopeduncular nucleus and substantia nigra, reminiscent of endogenous striatal neurons, which is not observed after grafting into adult hosts. M6 and M2-immunopositive cellular profiles from both the lateral and medial ganglionic eminences were observed to have migrated extensively away from the injection site, in contrast to the cerebellar precursors which remained clustered at the implantation site. Cells from the lateral ganglionic eminence were largely confined within the striatal complex where they developed striatal characteristics, displaying expression of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, whereas cells from the medial ganglionic eminence had migrated caudally along the internal capsule and were observed predominantly in the globus pallidus and thalamus, in addition to the striatum. The cells located outside the striatum were all DARPP-32 negative. The improved integration and increased projection capacity of the lateral ganglionic eminence precursors grafted into postnatal day 1 hosts gradually declined as the host advanced into later stages of development (postnatal day 7), and in postnatal day 21 hosts the grafted striatal precursors behaved similarly to grafts implanted into adult recipients. These results demonstrate the specific capacity of embryonic striatal progenitors to integrate into the developing basal ganglia circuitry during early postnatal development, and that the extent of neuronal and glial integration and graft host connectivity declines when the host has developed beyond the first postnatal week.

Shortcomings of current techniques used for the intracerebral transplantation of ventral mesencephalic dopamine neurons include low graft survival, high variability, considerable implantation trauma and suboptimal graft integration. In order to overcome these limitations, we have adopted a microtransplantation approach which allows precise and reproducible implantation of ventral mesencephalon cell suspensions at single or multiple sites with minimal trauma and improved survival and integration of the grafted neurons [Nikkhah et al. (1994) Brain Res. 633, 133-143]. The present study was undertaken to determine the influence of different grafting parameters as well as the time-course of development of micrografted dopaminergic neurons and to devise an optimal microtransplantation procedure in the rat Parkinson model, Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received four graft deposits of either 0.25, 0.5, 1.0 or 2.0 microliters along four injection tracts (150,000 cells/microliters) using either a glass capillary (o.d. 50-70 microns) or a regular cannula (o.d. 0.50 mm, metal cannula grafts). At one, two and 12 weeks postgrafting (capillary grafts) and at 12 weeks postgrafting (metal cannula grafts) dopamine neuron survival and graft volumes were measured and the implantation trauma assessed by glial fibrillary acidic protein expression. The results demonstrate that single deposits of 50,000-75,000 cells in 0.5 microliter, implanted with a glass capillary, provide the best environment both for dopaminergic and non-dopaminergic neuron survival. Grafts implanted with the glass capillary showed much weaker long-term glial fibrillary acidic protein expression along the injection tract and around the implants than was the case in grafts implanted with the thicker metal cannula. Optimal graft integration and minimal disturbances of host brain structures can reliably be achieved by small-sized implants (20,000-35,000 cells/deposit). Tyrosine hydroxylase-positive fiber outgrowth from micrografted dopaminergic neurons was seen not only in the surrounding caudate-putamen, but also along white matter tracts into the nucleus accumbens and the overlying cerebral cortex. Spreading of dopaminergic micrografts over multiple small deposits rather than increasing the volume of single grafts gave more extensive reinnervation of the entire host striatum. The micrografting technique provides a useful tool to improve graft-host interactions in the rat Parkinson model, and it allows more precise and reproducible quantitative studies on dopamine neuron survival and growth in intrastriatal ventral mesencephalon transplants. This technique should also be highly useful for the intracerebral implantation of cells derived from primary cultures or cell lines [Gage and Fisher (1991) Neuron 6, 1-12].