A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson's disease.

Neural progenitor cells potentially provide a limitless, on-demand source of cells for grafting into patients with Parkinson's disease (PD) if the signals needed to control their conversion into dopamine (DA) neurons could be identified. We have recently shown that cytokines which instruct cell division and differentiation within the hematopoeitic system may provide similar functions in the central nervous system. We have shown that mitotic progenitor cells can be isolated from embryonic rat mesencephalon and that these cells respond to a combination of interleukin-1, interleukin-11, leukemia inhibitory factor, and glial cell line-derived neurotrophic factor yielding a tyrosine hydroxylase-immunoreactive (THir) phenotype in 20-25% of total cells. In the present study, 24 clonal cell lines derived from single cells of mesencephalic proliferation spheres were examined for their response to the cytokine mixture. The clone yielding the highest percentage of THir neurons (98%) was selected for further study. This clone expressed several phenotypic characteristics of DA neurons and expression of Nurr1. The response to cytokines was stable for several passages and after cryopreservation for several months. When grafted into the striatum of DA-depleted rats, these cells attenuated rotational asymmetry to the same extent as freshly harvested embryonic DA neurons. These data demonstrate that mesencephalic progenitor cells can be clonally expanded in culture and differentiated in the presence of hematopoietic cytokines to yield enriched populations of DA neurons. When transplanted, these cells provide significant functional benefit in the rat model of PD.

Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval.

The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5-20%, and even poorer in grafts to the aged striatum. Grafted cells die in young adult rats during the first 4 days after implantation. The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals. We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation. At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats. This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting. THir neurons in grafts to the intact striatum possessed a significantly shorter "long axis" than their counterparts on the lesioned side. No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting. In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts.

Time course of apoptotic cell death within mesencephalic cell suspension grafts: implications for improving grafted dopamine neuron survival.

The vast majority ( congruent with 90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum. Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation. This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts. Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively. Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum. Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation. However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days. No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found. Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis.

Oligodendrocyte-type 2 astrocyte-derived trophic factors increase survival of developing dopamine neurons through the inhibition of apoptotic cell death.

Survival of embryonic dopamine (DA) neurons is extremely low (5-20%) following transplantation. Strategies to increase this survival are critical to the future of transplantation for Parkinson's disease. We demonstrate here that a factor(s) released from striatal oligodendrocyte-type 2 astrocytes (SO2A) greatly improves the survival and phenotype expression of mesencephalic DA neurons in culture while simultaneously decreasing the presence of apoptotic nuclear profiles, as detected by the TUNEL method and bisbenzamide/tyrosine hydroxylase double labeling. This SO2A-derived trophic factor(s) has minimal effects on glia and no effect on nondopaminergic mesencephalic neurons. The developmental period during which this SO2A trophic effect occurs (E14-18) coincides with the period when mesencephalic grafts are undergoing the highest rates of apoptosis, i.e., immediately following implantation. Therefore, SO2A-derived trophic factor(s) offers great potential for the augmentation of grafted DA neuron survival.

The distribution of reduced nicotinamide adenine dinucleotide phosphate-diaphorase in the leopard frog telencephalon and related projections.

The distribution of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was mapped histochemically in the forebrain of Rana pipiens, the leopard frog. Intense staining was observed which was strikingly restricted to certain nuclear groups and fiber tracts. The densest concentrations of NADPH-D stained cell bodies and fibers were observed in the granule layer of the accessory olfactory bulb and in the ventral aspect of the lateral pallium. Intense staining has also been reported in the presumed mammalian homologues of these regions. Less densely packed clusters of intensely stained neurons were found in the striatum, the anterior entopeduncular nucleus, the olfactory tubercle and the pars lateralis of the amygdala, whereas the preoptic region and the medial septum exhibited dense accumulations of lightly stained cells. Several fiber systems or terminal fields could be detected, including a ring of heavy staining which enclosed the striatum and an apparent terminal field in the lateral part of the medial pallium. A prominent compact tract, which may be homologous to a component of the stria terminalis of mammals, could be also followed from the ventral portion of the lateral pallium to the infundibular hypothalamus.

Dopamine agonists and stress produce different patterns of Fos-like immunoreactivity in the lateral habenula.

In rats treated systemically with either amphetamine, amfonelic acid or apomorphine, large numbers of cells displaying Fos-like immunoreactivity (FLI) could be seen in the lateral zone of the lateral habenula. The induction of FLI by amphetamine could be blocked either by pretreatment with haloperidol or by 6-hydroxydopamine lesions of ascending dopamine fibers at the level of the lateral hypothalamus. In contrast, a variety of stressors selectively induced FLI in the most medial portion of the lateral habenula. These findings support the concept of a functional differentiation of the medial and lateral regions of the lateral habenula and provide further evidence for involvement of the habenula in the circuitry of the basal ganglia.

Studies on the behavioral activation produced by stimulation of GABAB receptors in the median raphe nucleus.

Injections of the GABAB agonist baclofen into the median raphe nucleus (MR) resulted in marked hyperactivity and in increases in food and water intake by non-deprived animals. The locomotor effects of baclofen were stereospecific and could be antagonized by coinjection of the GABAB antagonist 2-hydroxysaclofen. Hyperactivity was produced by lower doses of baclofen, at shorter latencies, when the drug was injected into the MR than when it applied to the dorsal raphe nucleus (DR) or the ventral tegmental area (VTA). The locomotor response to intra-MR baclofen was unaltered in animals pretreated with the serotonin synthesis inhibitor p-chlorophenylalanine. Finally, intra-MR injections of baclofen produced a large increase in dopamine metabolism in the nucleus accumbens and striatum but failed to alter hippocampal or striatal serotonin metabolism. These findings suggest that baclofen may produce increases in activity and ingestive behavior as a result of an action on non-serotonergic cells in the MR.