Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.

Suspensional reaggregates of human foetal neocortex and tegmentum as objects of neurotransplantation.

Suspensional reaggregates were obtained from human neocortical and tegmental anlagen (7 weeks of gestation), using 0.1% tripsin solution, and cultivated in Medium 199. Suspensional reaggregates, formed after 2 days in vitro, were grafted into the Wistar rat striatum. Incipient stages of histogenesis in the reaggregates and their interaction with host brain were investigated using light and electron microscopy, with antibodies against vimentin, glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), ferritin, as well as lectin ricinus communis agglutinin (RCA). The reaggregates showed a low level of tissue organisation. An intermediate condition between suspension and the true tissue could be observed in them. These reaggregates had two evident features: a rather irregular cell arrangement (without parallel bundles of radial glia), and the presence of special intercellular junctions. Some cells made up fragments of neuroepithelial sheet in the form of true rosettes. The one-week-old grafts were integrated with the host brain as well as dissociated and contained host astrocytes. Degenerated cells and detritus appeared rarely. The data of this work let us conclude that the suspensional aggregate grafting combines some advantages of suspensional and solid grafting methods.

Combined mesencephalic and hypothalamic transplants reverse lesion-induced sexual behavior deficits in the male rat.

Studies of sexual behavior in rodent animal models have provided evidence about the relevant role played by the medial preoptic area of the anterior hypothalamus and the central tegmental field within the mesencephalon in the control of this behavior. Bilateral lesions of the anterior hypothalamus or central tegmental field as well as combined unilateral lesions of both these regions result in sexual behavior deficits. Studies using fetal hypothalamic transplants have been shown to reverse sexual behavior deficits induced either by lesions or aging. However, no previous study has evaluated the effect of combined homotopic transplants into both the anterior hypothalamus and the mesencephalon. In the present study male Wistar animals received two electrolytic lesions, one aimed at the ipsilateral medial preoptic area of the anterior hypothalamus and the other at the contralateral central tegmental field. Following these lesions, unilateral homotopic fetal hypothalamic and mesencephalic transplants were placed into the lesioned areas. Sexual behavior recovered gradually and by weeks 14-15 after transplantation, above 90% of animals with bilateral transplants showed mounts, intromissions, and ejaculations. Only animals with viable transplants located within both lesioned areas showed recovery. These results indicate that the behavioral deficits induced by combined unilateral lesions of hypothalamic and mesencephalic regions can be reversed by homotopic fetal transplants and that this recovery could be the result of the restoration of a behavioral relevant circuit between transplants and host brain nuclei separated by as much as 5 mm, which makes this an excellent model to study mechanisms underlying behavioral recovery after transplantation.

Nimodipine enhances growth and vascularization of neural grafts.

The potential of the calcium channel antagonist, nimodipine, to enhance vascularization and growth of neural grafts has been investigated. Four groups of rats received unilateral 6-OHDA lesions of the nigrostriatal pathway followed 16-23 days later by intrastriatal grafts of embryonic ventral mesencephalon. The grafts were derived from (i) Embryonic Day 14 embryos (group E14), (ii) Day 17 embryos (group E17), and (iii) Day 20 embryos (group E20), all implanted within 90 min of preparation, or (iv) Day 14 embryos with a 6-h delay prior to implantation (group E14/6H). Half the rats in each group received intragastric intubation with nimodipine daily for 14 days after transplantation surgery, whereas the other half received control intubations. The rats were killed by perfusion with formalin containing Indian ink 6 weeks after grafting. Nimodipine treatment enhanced the vascularization of the grafts in all four groups and induced a significant increase in the volume of grafts under the three suboptimal transplantation conditions (i.e., groups E17, E20, E14/6H). The results suggest that vascularization of graft tissue is an important determinant of survival and growth of neural transplants prepared by the dissociated cell suspension technique and show that vascular perfusion of grafts can be enhanced by nimodipine.

Intracerebral dopaminergic transplants are not activated by electrical footshock stress activating in situ mesocorticolimbic neurons.

Male rats received a dopaminergic implant aimed either at the nucleus accumbens or the ventral tegmental area (VTA) following 6-hydroxydopamine lesion of their mesocorticolimbic dopaminergic system. Exposure to electrical footshock stress 6 months later markedly activated the mesocorticolimbic neurons in control animals as shown by the increase of dihydroxyphenylacetic acid (DOPAC) levels both in the nucleus accumbens and the VTA. However, no stress-induced activation was seen for the grafted neurons, irrespective of the area of implantation. These results indicate the lack of reinnervation and modulation of the grafted dopaminergic neurons by one of the important afferent systems regulating the activity of endogenous mesencephalic dopaminergic neurons.

The formation of new neuronal circuit between transplanted nigral dopamine neurons and non-immunoreactive axon terminals in the host rat caudate nucleus.

Using immunoelectron microscopic techniques, whether or not host neuronal elements newly form synaptic contact with the grafted dopamine (DA) neurons in the caudate nucleus of the rat with unilateral lesion in the nigrostriatal DA pathway was examined. Tyrosine hydroxylase (TH) was used as a marker for DA-containing structures. Motor imbalances after the lesion and before or after the transplantation were assessed by the amount of circlings after the injection of Met-amphetamine. In animals which recovered well from motor imbalance, non-immunoreactive axon terminals made synaptic contact with grafted TH-positive cell bodies and their dendrites. Since the incidence of these synapses was quite low in poorly recovered animals, the formation of a new neuronal circuit may be one of the important bases for behavior recovery.

Axon terminal clustering in nigrostriatal double grafts.

Embryonic dopaminergic terminals of the ventral mesencephalon were cotransplanted with other immature neural tissues to the brain of newborn rats. Immunocytochemical localization of tyrosine hydroxylase in mature cografts with light and electron microscopy, demonstrated the existence of different axon terminal distributions in different grafts. These findings implicate the influence of local cell interactions in determining axon arborization.

Combined grafts of the ventral tegmental area and nucleus accumbens in oculo. Histochemical and electrophysiological characterization.

Dissection techniques and optimal donor stages have been established for constructing an isolated intraocular model of the ventral tegmental area (VTA)-accumbens system using intraocular sequential grafting. Single grafts including accumbens and VTA respectively survived and developed many organotypic features when taken from 15-17 day fetuses. Falck-Hillarp fluorescence histochemistry showed dopamine neurons and terminals in single VTA grafts, no or almost no catecholamine fibers in single accumbens grafts, and a well-developed VTA-accumbens dopamine pathway in combined grafts where cell bodies in the VTA part provided the accumbens part with a rich terminal network. A similar distribution was found using immunohistochemistry with antibodies directed against tyrosine hydroxylase. CCK-like immunoreactivity had a distribution that mimicked that of the catecholamine-containing system. Enkephalin-like immunoreactivity was found both in single VTA and in single accumbens pieces as well as in both parts of the double grafts. Cells with long-duration action potentials typical of dopamine neurons discharged at approximately 8 Hz in single VTA grafts and below 1 Hz in the VTA part of VTA-accumbens double grafts. Cells in the accumbens portion of double grafts had shorter action potential durations and fired at 10-20 Hz. Haloperidol increased discharge frequency in VTA neurons with long action potential durations while apomorphine reduced discharge markedly. Antidromic activation of putative dopamine neurons in the VTA part was obtained by electrical stimulation of the accumbens part. The indirect dopamine agonist + 3-methyl-phencyclidine slowed firing rates of neurons in the accumbens part of double grafts. Taken together, the histochemical and the electrophysiological data show that the intraocular VTA-accumbens system retains several of its normal structural and functional characteristics. It is proposed that the isolated VTA-accumbens projection can be used as a model to study the cellular mechanism of action of stimulant and opiate drugs of abuse.