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Human embryonic stem (hES) cell differentiation into dopamine neurons is considered a promising strategy for cell replacement therapy in Parkinson's disease, yet the functional properties of hES cell-derived dopamine neurons remain poorly defined. The objective of this study was to characterize intracellular calcium (Ca(2+)) and sub-plasma membrane cyclic AMP-signaling properties in hES cell-derived dopamine neurons. We found that hES cell-derived dopamine neurons and neural progenitors raised Ca(2+) from intra- and extracellular compartments in response to depolarization, glutamate, ATP, and dopamine D(2) receptor activation, while undifferentiated hES cells only mobilized Ca(2+) from intracellular stores in response to ATP and D(2) receptor-induced activation. Interestingly, we also found that hES cell-derived dopamine neurons in addition to primary ventral midbrain dopamine neurons were more prone to release Ca(2+) from intracellular stores than non-dopamine neurons following treatment with the neuropeptide neurotensin. Furthermore, hES cell-derived dopamine neurons showed cAMP elevations in response to forskolin and 3-isobutyl-methylxanthine, similar to primary dopamine neurons. Taken together, these results unravel the temporal sequence by which hES cells acquire Ca(2+) and cAMP signaling competence during dopamine differentiation.

Increasing evidence suggests that alpha-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two alpha-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1)+ precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca(2+)) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, alpha-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy.

Increasing evidence suggests that α-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two α-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1) precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca2+) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, α-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy. [ABSTRACT FROM AUTHOR]

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion- trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy. [ABSTRACT FROM AUTHOR]