Vasoactive intestinal peptide regulation of neuroblast mitosis and survival: role of cAMP.

The role of adenosine 3':5'-cyclic monophosphate (cAMP) in the regulation of neuroblast mitosis and survival by vasoactive intestinal peptide (VIP) was examined. VIP increased the cAMP content of cultured rat sympathetic neuroblasts. Further, cAMP stimulated DNA synthesis and survival in neuroblast cultures, replicating the effects of VIP. Thus, the VIP-cAMP signaling pathway may be involved in the regulation of neuronal development.

Role of voltage-sensitive calcium channels in mitogenic stimulation of neuroblasts.

The present study examines the role of Ca2+ in the regulation of sympathetic neuroblast mitosis. Employing a fully defined neuroblast culture system, we previously found that insulin growth factors (IGFs), depolarization and vasoactive intestinal peptide (VIP) regulated precursor mitosis. We now report that Ca2+ entry via voltage-sensitive channels was required for depolarization-stimulated mitogenesis. Ca2+ channel blockade with nitrendipine completely inhibited the increase in [3H]thymidine incorporation elicited by depolarizing stimuli including 30 mM KCl and the Na+ channel agonist veratridine. However, Ca2+ channel activity was not involved in the stimulation of DNA synthesis by IGFs or VIP. Thus, neuroblast mitosis may be regulated by multiple intracellular as well as extracellular signals.

Trophic mechanisms regulate mitotic neuronal precursors: role of vasoactive intestinal peptide (VIP).

While trophic support from targets depends on innervation, recent evidence suggests that local VIP promotes survival of sympathetic neuroblasts prior to target interactions, during the period of neurogenesis. Developmental studies now indicate that VIP expression peaks at embryonic day 15.5 (E15.5) in sympathetic ganglia in vivo, decreasing 3-fold by birth. The expression pattern in vivo paralleled the time course of ganglion neuroblast mitosis and peptide promotion of survival in culture. In contrast, nerve growth factor (NGF) exhibited a reciprocal trophic relationship, primarily supporting older neurons that were unresponsive to VIP. To define relationships of trophism to mitosis, serial time-lapse photography was employed to document the fate of neuroblasts produced by cytokinesis in vitro. In the absence of trophic factors, up to 80% of newly born cells died by 48 h, while virtually all neuroblasts survived in response to VIP plus NGF. In addition, trophic factors elicited multiple rounds of precursor division and an increase in absolute cell number, indicating that both trophic and mitogenic mechanisms contribute to proliferation. In aggregate, these observations suggest that VIP is expressed locally during a critical fetal period, providing trophic support to dividing ganglion neuroblasts prior to the action of target-derived NGF.

Neural stem and progenitor cells: a strategy for gene therapy and brain repair.

The damaged adult mammalian brain is incapable of significant structural self-repair. Although varying degrees of recovery from injury are possible, this is largely because of synaptic and functional plasticity rather than the frank regeneration of neural tissues. The lack of structural plasticity of the adult brain is partly because of its inability to generate new neurons, a limitation that has severely hindered the development of therapies for neurological injury or degeneration. However, a variety of experimental studies, as well as moderately successful clinical engraftment of fetal tissue into the adult parkinsonian brain, suggests that cell replacement is evolving as a valuable treatment modality. Neural stem cells, which are the self-renewing precursors of neurons and glia, have been isolated from both the embryonic and adult mammalian central nervous system. In the adult human brain, both neuronal and oligodendroglial precursors have been identified, and methods for their harvest and enrichment have been established. Neural precursors have several characteristics that make them ideal vectors for brain repair. They may be clonally expanded in tissue culture, providing a renewable supply of material for transplantation. Moreover, progenitors are ideal for genetic manipulation and may be engineered to express exogenous genes for neurotransmitters, neurotrophic factors, and metabolic enzymes. Thus, the persistence of neuronal precursors in the adult mammalian brain may permit us to design novel and effective strategies for central nervous system repair, by which we may yet challenge the irreparability of the structurally damaged adult nervous system.

Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts.

Although acute, millisecond-to-millisecond actions of neurotransmitters are well documented, diverse longer-term effects have been discovered only recently. Emerging evidence indicates that these signals regulate a variety of neuronal processes, from phenotypic expression to neurite outgrowth. Here we show that a single putative transmitter, vasoactive intestinal peptide, can exert multiple, long-term effects simultaneously: it stimulates mitosis, promotes neurite outgrowth and enhances survival of sympathetic neuron precursors in culture. As the peptide seems to be a normal presynaptic transmitter in the sympathetic system, synaptic transmission may exert hitherto unexpected effects.

Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells.

The adult mammalian forebrain harbors neuronal precursor cells in the subependymal zone (SZ). Neuronal progenitors also persist in the adult human SZ and have been cultured from epileptic temporal lobe. In the present study, we sought to identify these neural progenitors in situ, and to direct their expansion and neuronal differentiation in vitro. We prepared explants of adult human SZ, obtained from temporal lobe resections of refractory epileptics. The resultant cultures were treated with fibroblast growth factor-2 (FGF-2) for a week, with concurrent exposure to [3H]thymidine, then switched to media containing brain-derived neurotrophic factor (BDNF) for up to 2 months. Sporadic neuronal outgrowth, verified antigenically and physiologically, was observed from SZ cultures regardless of FGF-2/BDNF treatment; however, only FGF-2/BDNF-treated cultures exhibited profuse outgrowth, and these displayed neuronal survival as long as 9 weeks in vitro. In addition, cortical cultures derived from two brains generated microtubule-associated protein-2+ neurons, which incorporated [3H]thymidine and exhibited significant calcium increments to depolarization. In histological sections of the subependyma, both uncommitted and restricted progenitors, defined respectively by musashi and Hu protein expression, were identified. Thus, the adult human subependyma harbors neural progenitors, which are able to give rise to neurons whose numbers can be supported for prolonged periods in vitro.