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Background: Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development.

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, ␣-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 ؋ 10 6 compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10 6 compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.Cognitive impairment is a fundamental trademark of many CNS 2 diseases such as Alzheimer disease, schizophrenia, and attention deficit hyperactivity disorder. Unfortunately few pharmaceutical agents have proven to be efficacious in treating cognitive impairment. As such, the medical need for new therapeutic approaches for improving cognitive deficits has emerged as an important frontier in treating central nervous system diseases. Glutamate mediates the vast majority of excitatory neurotransmission in the CNS with post-synaptic signaling regulated by either ligand-gated cation channels (ionotropic) or G-protein-coupled receptors (metabotropic). Ionotropic ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are tetrameric aggregates composed of combinations of one or more of four subunits (GluR1-4) that can each bind a glutamate neurotransmitter molecule. Upon binding of glutamate, the associated ion channel pore opens, allowing cations to cross the membrane and depolarize the postsynaptic cell. Termination of the postsynaptic signal is driven primarily by the removal of glutamate from the synaptic cleft, reducing the occupancy of the glutamate binding site and subsequent channel closure (deactivation). Additionally, the conduction pathway will close in the continued presence of bound agonist as a result of conformational changes secondary to glutamate binding (desensitization).AMPA receptor pharmacology has identified agonists, competitive and non-competitive antagonists, and positive allosteric modulators (3). This latter class of pharmacologic agen...

Spiking activity in motor axons represents the final central coding for muscle contraction. Recurrent collaterals in spinal cord from these same axons are known to offer a negative feedback control of motor output via a class of interposed inhibitory interneurons. Here we demonstrate that, during noradrenergic drive, a previously unknown recurrent excitatory pathway is unmasked and expressed. These excitatory projections are shown to have broad bilateral actions within and between hindlimb spinal segments and can alter ongoing pattern-generating motor behaviors. Thus, motor output strength is controlled via central positive and negative feedback loops, undoubtedly to provide a greater flexibility and dynamic range of control. That this novel function is regulated by a descending neuromodulatory transmitter indicates a conditional recruitment during certain behavioral states as part of the central noradrenergic arousal apparatus.

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C‐fibre activation induces a long‐term potentiation (LTP) in the spinal flexion reflex in mammals, presumably to provide enhanced reflexive protection of damaged tissue from further injury. Descending monoaminergic pathways are thought to depress sensory input but may also amplify spinal reflexes; the mechanisms of this modulation within the spinal cord remain to be elucidated. We used electrical stimulation of primary afferents and recordings of motor output, in the rat lumbar spinal cord maintained in vitro, to demonstrate that serotonin is capable of inducing a long‐lasting increase in reflex strength at all ages examined (postnatal days 2–12). Pharmacological analyses indicated an essential requirement for activation of 5‐HT2C receptors while 5‐HT1A/1B, 5‐HT7 and 5‐HT2A receptor activation was not required. In addition, primary afferent‐evoked synaptic potentials recorded in a subpopulation of laminae III‐VI spinal neurons were similarly facilitated by 5‐HT. Thus, serotonin receptor‐evoked facilitatory actions are complex, and may involve alterations in neuronal properties at both motoneuronal and pre‐motoneuronal levels. This study provides the first demonstration of a descending transmitter producing a long‐lasting amplification in reflex strength, accomplished by activating a specific serotonin receptor subtype. It is suggested that brain modulatory systems regulate reflex pathways to function within an appropriate range of sensori‐motor gain, facilitating reflexes in behavioural situations requiring increased sensory responsiveness.

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC-derived NP cells. In NP cells, as well as NP-derived neurons, LIF reduced caspase-mediated apoptosis and reduced both spontaneous and H 2 O 2 -induced reactive oxygen species in culture. In vitro, NP cell proliferation and the yield of differentiated neurons were significantly higher in the presence of LIF. In NP cells, LIF enhanced cMyc phosphorylation, commonly associated with self-renewal/proliferation. Also, in differentiating NP cells LIF activated the phosphoinositide 3-kinase and signal transducer and activator of transcription 3 pathways, associated with cell survival and reduced apoptosis. When differentiated in LIF 1 media, neurite outgrowth and ERK1/2 phosphorylation were potentiated together with increased expression of gp130, a component of the LIF receptor complex. NP cells, pretreated in vitro with LIF, were effective in reducing infarct volume in a model of focal ischemic stroke but LIF did not lead to significantly improved initial NP cell survival over nontreated NP cells. Our results show that LIF signaling significantly promotes human NP cell proliferation, survival, and differentiation in vitro. Activated LIF signaling should be considered in cell culture expansion systems for future human NP cell-based therapeutic transplant studies.