Long-term Neuronal Survival Research Articles

Dissection, Plating, and Maintenance of Dorsal Horn Neuron Cultures: Figure 1.

INTRODUCTIONPrimary cultures of embryonic dorsal horn (DH) neurons can be prepared using mass cultures, microisland cultures, or macroisland cultures. In all cases, long-term survival of neurons is enhanced by the presence of astrocytes. After the feeder layer of astrocytes has become nearly or fully confluent, it is ready to receive freshly dissociated neurons. Embryonic dorsal root ganglion (DRG) neurons can also be added to the cultures if DRG/DH neuron cocultures are desired. These cultures are suitable for electrophysiological, molecular, and immunocytochemical studies. DH neurons, including both excitatory and inhibitory neurons, form synapses with themselves (autapses) and with each other. DRG neurons form synapses onto DH neurons. Finding synaptically connected pairs of neurons is most readily achieved when the cells are grown on microislands. This protocol describes the dissociation, plating, and maintenance of DH neuron cultures using a feeder layer of astrocytes.

NeuroD6 genomic signature bridging neuronal differentiation to survival via the molecular chaperone network

During neurogenesis, expression of the basic helix-loop-helix NeuroD6/Nex1/MATH-2 transcription factor parallels neuronal differentiation and is maintained in differentiated neurons in the adult brain. To dissect NeuroD6 differentiation properties further, we previously generated a NeuroD6-overexpressing stable PC12 cell line, PC12-ND6, which displays a neuronal phenotype characterized by spontaneous neuritogenesis, accelerated NGF-induced differentiation, and increased regenerative capacity. Furthermore, we reported that NeuroD6 promotes long-term neuronal survival upon serum deprivation. In this study, we identified the NeuroD6-mediated transcriptional regulatory pathways linking neuronal differentiation to survival, by conducting a genome-wide microarray analysis using PC12-ND6 cells and serum deprivation as a stress paradigm. Through a series of filtering steps and a gene-ontology analysis, we found that NeuroD6 promotes distinct but overlapping gene networks, consistent with the differentiation, regeneration, and survival properties of PC12-ND6 cells. By using a gene-set-enrichment analysis, we provide the first evidence of a compelling link between NeuroD6 and a set of heat shock proteins in the absence of stress, which may be instrumental in conferring stress tolerance on PC12-ND6 cells. Immunocytochemistry results showed that HSP27 and HSP70 interact with cytoskeletal elements, consistent with their roles in neuritogenesis and preserving cellular integrity. HSP70 also colocalizes with mitochondria located in the soma, growing neurites, and growth cones of PC12-ND6 cells prior to and upon stress stimulus, consistent with its neuroprotective functions. Collectively, our findings support the notion that NeuroD6 links neuronal differentiation to survival via the network of molecular chaperones and endows the cells with increased stress tolerance.

Isoflurane Differentially Affects Neurogenesis and Long-term Neurocognitive Function in 60-day-old and 7-day-old Rats

Anesthetic agents cause cell death in the developing rodent brain and long-term, mostly hippocampal-dependent, neurocognitive dysfunction. However, a causal link between these findings has not been shown. Postnatal hippocampal neurogenesis affects hippocampal function into adulthood; therefore, the authors tested the hypothesis that isoflurane affects long-term neurocognitive function via an effect on dentate gyrus neurogenesis. The S-phase marker 5-bromodeoxyuridine was administered at various times before, during, and after 4 h of isoflurane given to postnatal day (P)60 and P7 rats to assess dentate gyrus progenitor proliferation, early neuronal lineage selection, and long-term survival of new granule cell neurons. Fear conditioning and spatial reference memory was tested at various intervals from 2 weeks until 8 months after anesthesia. In P60 rats, isoflurane increased early neuronal differentiation as assessed by BrdU/NeuroD costaining, decreased progenitor proliferation for 1 day, and subsequently increased progenitor proliferation 5-10 days after anesthesia. In P7 rats, isoflurane did not induce neuronal lineage selection but decreased progenitor proliferation until at least 5 days after anesthesia. Isoflurane improved spatial reference memory of P60 rats long-term, but it caused a delayed-onset, progressive, persistent hippocampal deficit in P7 rats in fear conditioning and spatial reference memory tasks. The authors conclude that isoflurane differentially affects both neurogenesis and long-term neurocognitive function in P60 and P7 rats. Neurogenesis might mediate the long-term neurocognitive outcome after isoflurane at different ages.

Inhibition of Poly(ADP-Ribose) Polymerase Suppresses Inflammation and Promotes Recovery after Ischemic Injury

The brain inflammatory response induced by stroke contributes to cell death and impairs neurogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor NF-kappaB and required for NF-kappaB-mediated inflammatory responses. Here we evaluated PARP inhibition as a means of suppressing post-stroke inflammation and improving outcome after stroke. Rats were subjected to bilateral carotid occlusion-reperfusion, and treatment with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) was begun 48 h later. PJ34 was found to rapidly suppress the ischemia-induced microglial activation and astrogliosis. Behavioral tests performed 6 to 8 weeks after ischemia showed deficits in spatial memory and learning that were lessened by the PJ34 treatment. Immunohistochemical evaluation of hippocampus at 8 weeks after ischemia showed increased neuronal density in CA1 layer of PJ34-treated animals relative to vehicle-treated animals. Bromodeoxyuridine labeling showed formation of new neurons in hippocampal CA1 area in PJ34-treated animals, but not in vehicle-treated animals. Together, these results suggest that treatment with a PARP inhibitor for several days after ischemia enhances long-term neuronal survival and neurogenesis by reducing inflammation.

Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c

Summary ParagraphNeurons and cancer cells utilize glucose extensively, yet the precise advantage of this adaptation remains elusive. These two seemingly disparate cell types also exhibit an increased regulation of the apoptotic pathway, which allows for their long term survival1. Here we show that both neurons and cancer cells strictly inhibit cytochrome c-mediated apoptosis by a mechanism dependent on glucose metabolism. We report that the proapoptotic activity of cytochrome c is influenced by its redox state and that increases in Reactive Oxygen Species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c. In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH) generated as a result of glucose metabolism by the pentose phosphate pathway. These results uncover a striking similarity in apoptosis regulation between neurons and cancer cells and provide insight into an adaptive advantage offered by the Warburg effect for cancer cell evasion of apoptosis and for long-term neuronal survival.

Hypertonic Resuscitation Improves Neuronal and Behavioral Outcomes after Traumatic Brain Injury plus Hemorrhage

: Resuscitation with hypertonic saline or hypertonic saline plus l-arginine acutely improves cerebral blood flow after traumatic brain injury (TBI) followed by hemorrhagic hypotension. The authors investigated whether hypertonic saline or hypertonic l-arginine would improve long-term neuronal survival and behavioral outcomes 15 days after TBI and hemorrhagic hypotension. : Mean arterial pressure, arterial blood gases, pH, plasma glucose, hematocrit, and hemoglobin were measured in male Sprague-Dawley rats before and after moderate (2.0 atm) fluid percussion TBI. Rats were assigned to one of six groups: (1) sham TBI, (2) hemorrhage only, (3) TBI only, (4) TBI plus hemorrhage and resuscitation with 0.9% saline, (5) TBI plus hemorrhage and resuscitation with hypertonic saline (7.5%), or (6) TBI plus hemorrhage and resuscitation with l-arginine (100 mg/kg) in hypertonic saline. On postinjury days 1-5, vestibulomotor function was assessed using beam balance and beam walking tasks. On postinjury days 11-15, spatial memory function was assessed using the Morris water maze. After behavioral testing, neuronal counting was performed bilaterally on specific hippocampal regions. : Groups receiving hypertonic saline (P < 0.05, day 15 vs. day 11) or hypertonic l-arginine (P < 0.05, days 13-15 vs. day 11) showed improved performance over time on the Morris water maze, as well as significantly improved neuronal survival in the contralateral hippocampus (P < 0.05, hypertonic saline or hypertonic l-arginine vs. normal saline) compared with untreated TBI or normal saline-treated TBI plus hemorrhage groups. : Hypertonic saline and hypertonic l-arginine were both effective at promoting long-term neuronal survival and behavioral recovery. The slightly earlier improvement in Morris water maze performance in the hypertonic l-arginine group warrants further studies to determine whether higher doses of l-arginine provide additional improvement. This study supports the therapeutic benefits of hypertonic resuscitation after TBI plus hemorrhagic hypotension.

Antiapoptotic and Trophic Effects of Dominant-Negative Forms of Dual Leucine Zipper Kinase in Dopamine Neurons of the Substantia NigraIn Vivo

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model.

Delayed combinatorial treatment with flavopiridol and minocycline provides longer term protection for neuronal soma but not dendrites following global ischemia

We previously reported that delayed administration of the general cyclin-dependent kinase inhibitor flavopiridol following global ischemia provided transient neuroprotection and improved behavioral performance. However, it failed to provide longer term protection. In the present study, we investigate the ability of delayed flavopiridol in combination with delayed minocycline, another neuroprotectant to provide sustained protection following global ischemia. We report that a delayed combinatorial treatment of flavopiridol and minocycline provides synergistic protection both 2 and 10 weeks following ischemia. However, protected neurons in the hippocampal CA1 are synaptically impaired as assessed by electrophysio logical field potential recordings. This is likely because of the presence of degenerated processes in the CA1 even with combinatorial therapy. This indicates that while we have addressed one important pre-clinical parameter by dramatically improving long-term neuronal survival with delayed combinatorial therapy, the issue of synaptic preservation of protected neurons still exists. These results also highlight the important observation that protection does not always lead to proper function.

The Role of Rapid, Local, Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

Astrocytes enhance long‐term survival of cholinergic neurons differentiated from human fetal neural stem cells

Establishment of an in vitro model of human cholinergic neurons would be highly desirable for understanding and developing treatment for Alzheimer's and motoneuron diseases. Previously we reported that the combination of basic fibroblast growth factor (bFGF), heparin, and laminin directs human fetal neural stem cells to form cholinergic neurons. One problem, however, is that long-term in vitro survival of these cells is low. Our goal for this study was to determine whether astrocytes or their secreted factors enhance differentiation and survival of cholinergic neurons under long-term differentiation conditions. We demonstrate here that astrocytes or astrocyte conditioned media did not enhance cholinergic differentiation but did increase the long-term survival of differentiated human neural stem cells, particularly cholinergic neurons. We further show that astrocytes protected long-term-differentiated cells from apoptotic cell death, which is at least partially mediated by astrocyte-secreted bFGF. Our findings indicate that long-term survival of human stem cell-derived cholinergic neurons requires trophic factors from nonneuronal cells. This data may provide insights into the development of an in vitro model of long-term cultured human cholinergic neurons useful for understanding of the mechanisms of cholinergic differentiation and developing treatments for neurological diseases.

Neurotrophins improve synaptic transmission in the adult rodent diaphragm

Neurotrophins are usually viewed as secreted proteins that control long-term survival and differentiation of neurons. However, recent studies have established that among the most important functions of neurotrophins is their capacity to regulate synaptic functions and plasticity. When altering synaptic function, neurotrophins are able to produce two types of outcomes, an immediate effect on synaptic transmission and long-term control of synaptic structure and function. The first effect occurs within seconds or minutes after the neurotrophic factor has been applied and usually involves acute modification of synaptic transmission. The second effect takes hours and days, as protein synthesis is required to complete the structural changes. Neurotrophins and their receptors are expressed within the neuromuscular system, making these agents ideal candidates for the short-and long-term regulation of skeletal muscle function. For instance, neurotrophins can alter neuromuscular function acutely, by modulating the amount of neurotransmitter released with each nerve impulse, or chronically, by changing postsynaptic properties or the content and size of synaptic vesicles. It is obvious that the effects of neurotrophins depend on the specific neurotrophin involved (four neurotrophins have been found in mammals; these are nerve growth factor, brain-derived neurotrophic factor, and neurotrophins-3 and-4) and on the specific synapse being studied. Growing evidence highlights the role of neurotrophins in the development and function of neuromuscular synapses. This review will examine the role of neurotrophins in the regulation of neuromuscular transmission.

Neurogenesis in the adult rat piriform cortex

Multipotent neural precursors have been suggested to exist in many parts of the adult mammalian brain. In the present study, we characterized the neurogenic potential in the piriform cortex of adult rats. Proliferation rates as detected by 5'-bromodeoxyuridine-labeling proved to be low when compared with the major neurogenic brain regions (i.e. the hippocampus and the subventricular zone). 5'-Bromodeoxyuridine/NeuN-labeling in accordance with doublecortin, polysialylated neural cell adhesion molecule, and TUC-4-labeling indicated that neuronal differentiation of newborn cells occurs predominantly in layer II of the piriform cortex. Many of the cells exhibited a pyramidal cell morphology. The lack of 5'-bromodeoxyuridine/NeuN-labeled cells 12 weeks after 5'-bromodeoxyuridine administration argued against long-term survival of newborn neurons in the piriform cortex.

Neurons survive with NCS-1

A calcium sensor helps save damaged neurons, as reported by Nakamura et al. (page 1081). Calcium is a favorite messenger of neurons—it regulates neurotransmission, signal transduction, and synapse plasticity. The calcium-binding protein NCS-1 translates increased intracellular calcium levels into many of these downstream outcomes. The new results show that NCS-1 also activates antiapoptotic pathways in neurons. Neuronal apoptosis can result from injury or infection. Neurons often survive these insults, however, thanks to activation of the Akt survival pathway by neurotrophic factors such as GDNF. The authors show that this effect requires NCS-1, which is induced by GDNF. When activated by calcium (whose cytoplasmic levels probably increase upon injury), NCS-1 is known to activate phosphoinositide kinases that produce Akt substrates, thus jumpstarting the survival pathway. Loss of NCS-1 hastened cell death in injured rat brains. It also hampered the long-term survival of neurons under normal culture conditions, suggesting that low levels are required even in the absence of injury. As injury strongly increases the levels of NCS-1, its overexpression did not improve survival further. But in diseases in which neuronal survival and regeneration is impaired, boosting NCS-1 activity might have benefical effects.

Constitutively expressed catalytic proteasomal subunits are up‐regulated during neuronal differentiation and required for axon initiation, elongation and maintenance

Inhibition of the proteasome by lactacystin, a specific blocker of the catalytic beta-subunits, results in transient neurite outgrowth by neuronal cell lines. Vice versa, as demonstrated in this study, treatment of pheochromocytoma (PC12) cells with nerve growth factor (NGF) or other differentiating agents reduces proteasomal activity. This is accompanied by an increase in mRNA and protein levels of the catalytically active subunits beta1, beta2 and beta5, but not of their inducible counterparts, indicating changes in subunit composition of the proteasome during neuronal differentiation. In contrast to neuronal cell lines, however, pre-treatment of primary neurons with proteasome inhibitors completely prevents axon formation, and lower concentrations of lactacystin (0.5-5 microm) significantly reduce axonal elongation and branching in vitro. Furthermore, established axonal networks degenerate rapidly and long-term survival of peripheral neurons is impaired in the presence of proteasome inhibitors. Axonal pathology is reminiscent of the morphological changes observed in neurodegenerative disorders and supports a crucial role of the constitutive catalytic subunits in axon initiation, maintenance and regeneration.

The Alpha2-Adrenoceptor Antagonist Dexefaroxan Enhances Hippocampal Neurogenesis by Increasing the Survival and Differentiation of New Granule Cells

The generation of new neurons in the hippocampus is a dynamic process regulated by environmental, endocrine, and pharmacological factors. Since enhancement of hippocampal neurogenesis has been associated with learning and memory, and the locus coeruleus-noradrenergic system has been shown to modulate these cognitive functions, we hypothesized that activation of noradrenergic neurotransmission might enhance neurogenesis in the adult hippocampus. To test this hypothesis in vivo, we induced the release of noradrenaline in the hippocampus by blocking presynaptic inhibitory autoreceptors with the selective alpha2-adrenoceptor antagonist dexefaroxan. Confocal microscopy showed that noradrenergic afferents make contact with proliferating and differentiating cells, suggesting a direct noradrenergic influence on neurogenesis. Chronic systemic treatment of rats with dexefaroxan did not affect cell proliferation per se in the dentate gyrus (as monitored by bromodeoxyuridine-labeling), but promoted the long-term survival of newborn neurons by reducing apoptosis. Dexefaroxan treatment also enhanced the number and complexity of the dendritic arborizations of polysialated neural cell adhesion molecule-positive neurons. The trophic effects of dexefaroxan on newborn cells might involve an increase in brain-derived neurotrophic factor, which was upregulated in afferent noradrenergic fiber projection areas and in neurons in the granule cell layer. By promoting the survival of new endogenously formed neurons, dexefaroxan treatment represents a potential therapeutic strategy for maintaining adult neurogenesis in neurodegenerative conditions, such as Alzheimer's disease, that affect the hippocampus.

Neuronal degeneration and iNOS expression in experimental brain contusion following treatment with colchicine, dexamethasone, tirilazad mesylate and nimodipine

The pathophysiological mechanisms of secondary neurological injury after traumatic brain injury are complex. Post-traumatic biochemical reactions include parenchymal inflammation, free radical production, increased intracellular calcium and lipid peroxidation and nitric oxide production. The relative importance of each mechanism is unknown in brain contusions. This study was undertaken to investigate protection by the neuroprotective and/or anti-inflammatory drugs that have different putative mechanisms of action: colchicine, dexamethasone, tirilazad mesylate and nimodipine. A brain contusion was produced using a weight-drop model in rats. The animals were treated with either one of the drugs at previously defined relevant dosage or control. Fluoro-Jade labelling, TUNEL-staining and immunohisto-chemistry were used to study neuronal degeneration, cellular apoptosis and iNOS expression. In addition, the number of surviving neurons after 14 days was determined. The number of degenerating neurons was significantly reduced in all treatment groups at 24 hours while the total number of apoptotic cells including inflammatory cells and glia was unchanged. iNOS-expression was reduced in all treatment groups at 24 hours but not later. Only colchicine and tirilazad mesylate significantly enhanced neuronal survival at 14 days after injury. The findings underscored that an early neuroprotective effect does not necessarily lead to increased long-term neuronal survival. The absence of a significant long-term effect with nimodipine and dexamethasone agrees with clinical studies. Colchicine with an anti-macrophage/anti-inflammatory activity and the free radical scavenger tirilazad mesylate were effective for amelioration of experimental contusion with moderate energy transfer. Early neuroprotection may to some extent target iNOS via different pathways since all tested drugs affected both iNOS expression and neuronal degeneration.

Influence of Supporting Cells on Neuronal Degeneration After Hair Cell Loss

In sensorineural hearing loss, hair cell loss is often followed by loss of cochlear nerve fibers, which can continue for years after the insult. The degree and time course of neuronal loss varies, but the reasons for this variation are unclear. The present study addresses this issue with a quantitative analysis of hair cell, supporting cell, and neuronal survival in animals with long-term survival of up to 5.5 years from two types of drug-induced hair cell loss: aminoglycoside antibiotics and platinum-containing chemotherapeutics. To complement the analysis of the effects of organ of Corti damage on neuronal survival, cases of primary neuronal degeneration, via auditory nerve section, are also assessed. Analysis shows that (1) long-term neuronal survival is enhanced when supporting cells in the inner hair cell (IHC) area remain intact; (2) after hair cell loss, the time course of neuronal loss is slower in the apex than in the base; (3) primary loss of cochlear nerve fibers does not lead to secondary degeneration of sensory cells or supporting cells in the organ of Corti; and (4) after auditory nerve section, there can be a massive reinnervation of the IHC region, especially in the apex. Results are consistent with the idea that supporting cells participate in the regulation of neuronal survival and neuronal sprouting in the organ of Corti.

Pig xenografts to the immunocompetent rat brain: Survival rates using distinct neurotoxic lesions in the nigrostriatal pathway and two rat strains

Porcine foetal neurons for xenotransplantation in Parkinson's disease (PD) is an alternative source to human fetuses. One of the obstacles facing brain xenotransplantation is the existence of an immune response, which prevents long-term graft survival. Experimental results concerning the survival time of porcine foetal neurons implanted into the brain of immunocompetent rats have been quite different from one study to another, suggesting an effect on graft survival of uncontrolled experimental parameters. To identify such parameters, we have first analyzed the survival of porcine foetal nigral neurons at 5 and 10 weeks after implantation into the striatum of immunocompetent rats having different types of brain lesion affecting cells (quinolinic acid) or projections to the striatum (MPP +, 6-OHDA). In a second experiment, graft survival was analyzed in two strains of recipient rats (female Sprague–Dawley and male Lewis rats) in conditions of ipsilateral dopaminergic denervation using 6-OHDA. The characteristics of surviving grafts were assessed by measuring the graft volume, the number of TH + neurons, the size of TH + neurons soma, and CD5 + cell infiltration. Long-term survival (≥10 weeks) of porcine neurons could be observed in all experimental models. However, there was no significant difference in graft survival rates and characteristics of the surviving grafts between the lesioned groups, or between Sprague–Dawley and Lewis rats. Altogether, results were highly variable within groups of grafts exposed to similar experimental procedures at both 5 and 10 weeks post-grafting. We conclude that the distinct neurotoxins and host rat strains used in our experimental design are not major factors influencing the rejection time-course of primary neural xenografts.

Long distance selective fiber outgrowth of transplanted hNT neurons in white matter tracts of the adult rat brain

Terminally differentiated neurons derived from a human teratocarcinoma cell line (NT2N or hNT neurons) are promising as a cell source for transplantation, as they have been shown to be safe for transplantation in humans. We have shown previously that hNT neurons can express a catecholaminergic phenotype in a rat Parkinson model. In this study, we investigated the long-term survival and ability of hNT neurons to express tyrosine hydroxylase and reconstruct the dopamine-denervated nigrostriatal pathway. Hemiparkinsonian rats received grafts of 400,000 viable hNT neurons into each of the denervated striatum and substantia nigra. Robust hNT grafts were detected up to 24 weeks posttransplantation, although few cells expressed tyrosine hydroxylase. Many hNT fibers were often associated with ipsilateral and contralateral white matter tracts--corpus callosum, rostral migratory stream, optic tract, and external capsule. Fewer fibers were associated with the superior cerebellar peduncle, medial lemniscus, and nigrostriatal pathway. Axons also projected into the frontal cortex and extended parallel to the surface of the brain in the superficial cortical layers. These pathways were seen in all grafted animals, suggesting that specific guidance cues exist in the adult brain governing hNT fiber outgrowth. Injured adult axons and transplanted embryonic neuronal axons rarely extend for such distances in the adult nervous system. We propose that elucidating the factors promoting and guiding hNT axonal outgrowth could provide important clues to enhancing regeneration and target reinnervation in the adult brain, two factors of critical importance for cell restoration strategies aimed at brain repair.

Dazed gene is necessary for late cell type development and retinal cell maintenance in the zebrafish retina

Several molecules, such as growth factors and neurotrophic factors, are required both for the differentiation of specific retinal cell types and the long-term cell survival of all retinal neurons. As diffusible factors, these molecules act non-cell-autonomously. Here, we describe the loss of function phenotype for dazed (dzd), a gene that acts cell-autonomously for retinal cell survival and affects the differentiation of rod photoreceptors and the Muller glia. By 3 days after fertilization, dazed mutant embryos have small eyes and slight heart edema. Acridine orange staining indicated a significant degree of retinal cell death occurring by 48 hr after fertilization, and histological analysis revealed that dying cells were found in the inner and outer nuclear layers and near the marginal zones. Although molecular and morphological differentiation of the inner retina and cone photoreceptors occurred, rod photoreceptors failed to differentiate beyond a small patch in the ventral retina and rod precursors failed to respond to exogenously added retinoic acid, which normally potentiated rod differentiation. Mosaic analysis indicated that the dazed gene acts cell-autonomously for rod production and cell survival, as dazed clones failed to produce rods outside the ventral patch and dazed cells were not maintained in wild-type hosts. Raising mutants under constant light resulted in severe retinal degeneration, whereas raising embryos under constant darkness did not provide any additional protection from cell death. Behavioral analysis showed that a subpopulation of adult fish that were heterozygous for the dazed mutation had elevated visual thresholds and were night blind, suggesting that dazed may also be required for long-term dim-light vision. Taken together, our studies suggest a role for the dazed gene in rod and Muller cell development and overall retinal cell survival and maintenance.