Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain

While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain

Determinants of Functional Coupling between Astrocytes and Respiratory Neurons in the Pre-Bötzinger Complex

Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive

VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation

The “One neuron-one neurotransmitter” concept has been challenged frequently during the last three decades, and the coexistence of neurotransmitters in individual neurons is now regarded as a common phenomenon. The functional significance of neurotransmitter coexistence is, however, less well understood. Several studies have shown that a subpopulation of dopamine (DA) neurons in the ventral tegmental area (VTA) expresses the vesicular glutamate transporter 2 (VGLUT2) and has been suggested to use glutamate as a cotransmitter. The VTA dopamine neurons project to limbic structures including the nucleus accumbens, and are involved in mediating the motivational and locomotor activating effects of psychostimulants. To determine the functional role of glutamate cotransmission by these neurons, we deleted VGLUT2 in DA neurons by using a conditional gene-targeting approach in mice. A DAT-Cre/Vglut2Lox mouse line (Vglut2f/f;DAT-Cre mice) was produced and analyzed by in vivo amperometry as well as by several behavioral paradigms. Although basal motor function was normal in the Vglut2f/f;DAT-Cre mice, their risk-taking behavior was altered. Interestingly, in both home-cage and novel environments, the gene targeted mice showed a greatly blunted locomotor response to the psychostimulant amphetamine, which acts via the midbrain DA system. Our results show that VGLUT2 expression in DA neurons is required for normal emotional reactivity as well as for psychostimulant-mediated behavioral activation

SLC10A4 is a vesicular amine-associated transporter modulating dopamine homeostasis.

BACKGROUND: The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis. METHODS: We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine- regulated behaviors. RESULTS: We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice over-expressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine. CONCLUSIONS: Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders

Roadmap for the development of the University of North Carolina at Chapel Hill Genitourinary OncoLogy Database—UNC GOLD

BACKGROUND: The management of genitourinary malignancies requires a multidisciplinary care team composed of urologists, medical oncologists and radiation oncologists. A genitourinary (GU) oncology clinical database is an invaluable resource for patient care and research. Although electronic medical records provide a single web-based record used for clinical care, billing and scheduling, information is typically stored in a discipline-specific manner and data extraction is often not applicable to a research setting. A GU oncology database may be used for the development of multidisciplinary treatment plans, analysis of disease-specific practice patterns, and identification of patients for research studies. Despite the potential utility, there are many important considerations that must be addressed when developing and implementing a discipline-specific database. METHODS AND MATERIALS: The creation of the GU oncology database including prostate, bladder and kidney cancers with the identification of necessary variables was facilitated by meetings of stakeholders in medical oncology, urology, and radiation oncology at the University of North Carolina (UNC) at Chapel Hill with a template data dictionary provided by the Department of Urologic Surgery at Vanderbilt University Medical Center. Utilizing Research Electronic Data Capture (REDCap, version 4.14.5), the UNC Genitourinary OncoLogy Database (UNC GOLD) was designed and implemented. RESULTS: The process of designing and implementing a discipline-specific clinical database requires many important considerations. The primary consideration is determining the relationship between the database and the Institutional Review Board (IRB) given the potential applications for both clinical and research uses. Several other necessary steps include ensuring information technology security and federal regulation compliance; determination of a core complete data set; creation of standard operating procedures; standardizing entry of free text fields; use of data exports, queries, and de-identification strategies; inclusion of individual investigators’ data; and strategies for prioritizing specific projects and data entry. CONCLUSIONS: A discipline-specific database requires a buy-in from all stakeholders, meticulous development, and data entry resources in order to generate a unique platform for housing information that may be used for clinical care and research with IRB approval. The steps and issues identified in the development of UNC GOLD provide a process map for others interested in developing a GU oncology database

Selective regulation of nuclear orphan receptors 4A by adenosine receptor subtypes in human mast cells

Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A2A (A2AAR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A2B and A3) and that A2AAR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A2AAR-silencing amplified NR4A induction by NECA. Interestingly, a similar A2AAR modulatory effect was observed on ERK1/2 phosphorylation because A2AAR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A2AAR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A2AAR activation, not only in the context of adenosine but of other mast cell activating stimuli as well

Electrospray mass spectrometry for the direct accurate mass measurement of ligands in complex with the retinoid X receptor α ligand binding domain

Accurate mass measurements are often used in the structural determination of unknown compounds of low molecular mass (i.e., below ∼500 Da). Recently, it has been shown that accurate mass measurements also can be made on small denatured proteins (i.e., Mr, ∼17,000) to confirm their amino acid composition and identify the presence of isoforms. In the current report, we present nondenaturing electrospray (ES) mass spectrometry data on the direct accurate mass measurement of ligands in complex with the retinoid X receptor ligand binding domain (RXR LBD; Mr 31,370.92). Average mass errors were below 0.198 Da, 6.3 ppm (standard deviation [SD], 0.146; n = 10) for low-affinity fatty acid agonists analyzed in complex with the RXR LBD. Protein consumption was less than 15 pmol, with fatty acid ligands present at concentrations corresponding to their median effective concentration value (low micromolar, determined in transfection assays). Although determination of fatty acid mass was only sufficiently accurate to give nominal mass values, measurements were of sufficient accuracy to assign fatty acid chain length, degree of unsaturation, or cyclization. Using 17β-estradiol as a control, the ability to observe specific ligand binding is shown for both high- and low-affinity RXRα agonists. In addition, binding of a novel synthetic receptor agonist XCT0315908 to the RXRα LBD is reported. This compound showed a high degree of complex formation, and the receptor–ligand complex could be mass measured with an average mass error of −0.024 Da, 0.8 ppm (SD, 0.092; n = 9). Thus, specific binding of both nanomolar and micromolar affinity ligands to a nuclear receptor LBD can be directly observed using nondenaturing ES mass spectrometry and accurate mass measurements additionally can be made on intact complexes in the same experiment. This methodology also is applicable when ligands are present as components of mixtures