Ontogeny of substantia nigra dopamine neurons.

Understanding the ontogeny of A9 dopamine (DA) neurons is critical not only to determining basic developmental events that facilitate the emergence of the substantia nigra pars compacta (SNc) but also to the extraction and de novo generation of DA neurons as a potential cell therapy for Parkinson's disease. Recent research has identified a precise window for DA cell birth (differentiation) in the ventral mesencephalon (VM) as well as a number of factors that may facilitate this process. However, application of these factors in vitro has had limited success in specifying a dopaminergic cell fate from undifferentiated cells, suggesting that other cell/molecular signals may as yet remain undiscovered. To resolve this, current work seeks to identify particularly potent and novel DA neuron differentiation factors within the developing VM specifically at the moment of ontogeny. Through such (past and present) studies, a catalog of proteins that play a pivotal role in the generation of nigral DA neurons during normal CNS development has begun to emerge. In the future, it will be crucial to continue to evaluate the critical developmental window where DA neuron ontogeny occurs, not only to facilitate our potential to protect these cells from degeneration in the adult brain but also to mimic the developmental environment in a way that enhances our ability to generate these cells anew either in vitro or in vivo. Here we review our present understanding of factors that are thought to be involved in the emergence of the A9 dopamine neuron group from the ventral mesencephalon.

Ascorbic acid increases the number of dopamine neurons in vitro and in transplants to the 6-OHDA-lesioned rat brain.

The inadequate survival of dopamine neurons following intracerebral transplantation is in part attributed to the generation of reactive oxygen species and subsequent oxidative stress. To address this, we investigated whether the antioxidant ascorbic acid (vitamin C) had any effect on the yields of dopamine neurons derived from E14 rat ventral mesencephalic cells in vitro and in grafts. Following in vitro differentiation in medium containing ascorbic acid at concentrations ranging from 20 to 100 microM, significantly more neurons were immunopositive for the marker of mesencephalic dopamine neurons, tyrosine hydroxylase (TH), when compared to standard differentiation conditions containing no ascorbic acid. Mesencephalic cell suspensions supplemented with 100 microM ascorbic acid were also transplanted into unilateral 6-OHDA-lesioned rats and behavioral rotation was assessed at 2, 4, and 6 weeks posttransplantation. Grafts pretreated with ascorbic acid contained significantly more surviving dopamine neurons compared to nontreated grafts. However, no significant difference in rotation score was observed, with both groups showing a reversal and overcompensation of rotational bias. In addition, no evidence of neurogenesis of nigral dopamine neurons was observed in transplant groups. While the increased number of dopamine neurons observed in our study following ascorbic acid treatment may reflect a selective survival effect, our in vitro results suggest that ascorbic acid may act to increase the number dopamine neurons, both in culture and following transplantation, by stimulating dopaminergic differentiation of neural precursors from the fetal ventral mesencephalon.

The role of pretraining on skilled forelimb use in an animal model of Huntington's disease.

After a unilateral striatal lesion, animals have generally been seen to have a bilateral impairment in paw reaching, with the contralateral paw being more affected. However, most studies to date have not used a pretraining paradigm to assess maximal capacity for paw reaching, to compare with any lesion-induced loss. This study compared animals that were pretrained with naive animals in their ability to paw reach after a striatal lesion, to address the role of the striatum in either acquisition or execution of this motor task. All lesioned animals showed a significant decrease in reaching ability with their contralateral paw compared with the ipsilateral paw. Pretrained lesioned animals showed a clear lesion deficit with the contralateral paw immediately after lesion, and no impairment whatsoever with the ipsilateral paw. Naive lesioned animals showed delayed acquisition of the task with both paws, possibly due to postural deficits, and a lasting deficit on the contralateral side. The variability of performance between animals was higher in the naive lesioned group. These results suggest that animals should be pretrained on the staircase task prior to lesion to enable maximum sensitivity in detecting both loss and recovery of function of skilled forelimb use.

Embryonic neural progenitor cells: the effects of species, region, and culture conditions on long-term proliferation and neuronal differentiation.

One of the major obstacles to the use of neural stem/progenitor cells in neuronal replacement therapy is the limited ability of these cells to generate sufficient numbers of specific neuronal phenotypes either in the culture dish or after transplantation in animal models of neurodegenerative disease. It is not yet fully understood whether embryonic neural stem and progenitor cells show species-specific or regional identities, or if current culture paradigms select for a particular subset of stem cells/progenitors with similar proliferation and differentiation capacities. To investigate this issue, we isolated embryonic neural progenitors derived from the developing rat and mouse central nervous system for in vitro culture to assess the regional, species-specific, and temporal effects on both cell proliferation and generation of neurons. Neurosphere cultures were derived from E13-15 mouse or rat developing striatum (medial, lateral, or whole ganglionic eminence), ventral mesencephalon, and cortex. We compared basic fibroblast growth factor and epidermal growth factor for their influence on cell proliferation and neuronal differentiation under defined differentiation paradigms. Seeding density and conditioned media were also tested for their effects on maintenance of cell proliferation over protracted time periods. Results showed that embryonic neural stem/progenitor cells maintained defined patterns of proliferation and neuronal differentiation, with both declining with time in vitro. Proliferation rate was more dependent on species and region than the neurotrophins or conditions used for culture. These results suggest that the appropriate selection of embryonic neural stem cells and culture conditions may be crucial for the optimization of their neurogenic potential.

Neural transplantation: restoring complex circuitry in the striatum.

During the last 30 years, the promise of neural transplantation as a therapeutic strategy for neurodegenerative disease has been slowly recognised. Across the world, clinical transplants of embryonic primary dopamine neurones have been shown to ameliorate some of the motor deficits in Parkinson s disease (PD) patients, and more recently, systematic clinical trials have been initiated for the replacement of striatal projection neurones lost in Huntington's disease (HD). Clinical transplantation as a prospective therapy for HD poses a particular set of difficulties. The hallmarks of this neurodegenerative disease include extensive loss of medium spiny long-distance projection neurones of the caudate and putamen, affecting downstream target nuclei, the globus pallidus and substantia nigra, leading to dysregulation of motor control. In addition, extensive loss of cortical neurones that form the afferent systems to the basal ganglia leads to widespread cognitive decline. If transplantation therapy is to succeed in replacing degenerating neurones in HD and reinstating controlled function of complex basal gan-glia circuitry, the new neurones must be able to develop specific long-distance projections that can form accurate and functional connections with neurones in precise target regions. Our ongoing studies are aimed at addressing how we can improve the function of striatal transplants, in particular to optimise the reformation of precise long-distance connections and to re-establish normal motor and cognitive function. In particular, we have investigated optimal requirements for embryonic primary tissue to achieve these aims, and also the potential of other cell sources to provide long-distance projection neurones and reconnect complex circuitry. This review describes current progress of experiments to optimise the reconstruction of neuronal circuitry using primary embryonic tissue transplants, as well as our current initiatives to use neural stem cells or precursors to replace long distance projection neurones in the degenerating basal ganglia.

EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum.

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.

Transplanted neuroblasts differentiate appropriately into projection neurons with correct neurotransmitter and receptor phenotype in neocortex undergoing targeted projection neuron degeneration.

Reconstruction of complex neocortical and other CNS circuitry may be possible via transplantation of appropriate neural precursors, guided by cellular and molecular controls. Although cellular repopulation and complex circuitry repair may make possible new avenues of treatment for degenerative, developmental, or acquired CNS diseases, functional integration may depend critically on specificity of neuronal synaptic integration and appropriate neurotransmitter/receptor phenotype. The current study investigated neurotransmitter and receptor phenotypes of newly incorporated neurons after transplantation in regions of targeted neuronal degeneration of cortical callosal projection neurons (CPNs). Donor neuroblasts were compared to the population of normal endogenous CPNs in their expression of appropriate neurotransmitters (glutamate, aspartate, and GABA) and receptors (kainate-R, AMPA-R, NMDA-R. and GABA-R), and the time course over which this phenotype developed after transplantation. Transplanted immature neuroblasts from embryonic day 17 (E17) primary somatosensory (S1) cortex migrated to cortical layers undergoing degeneration, differentiated to a mature CPN phenotype, and received synaptic input from other neurons. In addition, 23.1 +/- 13.6% of the donor-derived neurons extended appropriate long-distance callosal projections to the contralateral S1 cortex. The percentage of donor-derived neurons expressing appropriate neurotransmitters and receptors showed a steady increase with time, reaching numbers equivalent to adult endogenous CPNs by 4-16 weeks after transplantation. These results suggest that previously demonstrated changes in gene expression induced by synchronous apoptotic degeneration of adult CPNs create a cellular and molecular environment that is both permissive and instructive for the specific and appropriate maturation of transplanted neuroblasts. These experiments demonstrate, for the first time, that newly repopulating neurons can undergo directed differentiation with high fidelity of their neurotransmitter and receptor phenotype, toward reconstruction of complex CNS circuitry.