Kieran W. McDermott

Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin‐positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self‐renewing radial cells, distinct from an earlier pseudo‐stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP‐positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.

Sexually dimorphic effects of maternal separation stress on corticotrophin-releasing factor and vasopressin systems in the adult rat brain

Neonatal maternal separation has been widely used to model the well‐established causal relationship between stress in early life and the later development of depression. As corticotrophin‐releasing factor (CRF) and vasopressin (AVP) have been implicated in depression, we aimed to determine the long‐term effects of maternal separation stress on these neuropeptide systems, and also to explore whether these effects are gender‐dependent. Immunohistochemical staining of CRF, AVP and c‐Fos was used to assess whether these neuropeptide systems were affected following an acute swim stress in male and female maternally separated rats.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 μM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 μs in air-saturated to 57 μs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Role of radial glia in cytogenesis, patterning and boundary formation in the developing spinal cord

Radial glial fibres provide a transient scaffold and impose constraints in the developing central nervous system (CNS) that facilitate cell migration and axon growth. Recent reports have raised doubts about the distinction between radial glia and precursor cells by demonstrating that radial glia are themselves neuronal progenitor cells in the developing cortex, indicating a dual role for radial glia in both neurogenesis and migration guidance. Radial glia shift toward exclusive generation of astrocytes after neurogenesis has ceased. Radial progenitor cell differentiation and lineage relationships in CNS development are complex processes depending on genetic programming, cell–cell interaction and microenvironmental factors. In the spinal cord, radial cells that arise directly from the neuroepithelium have been identified. At least in the spinal cord, these radial cells appear to be the precursors to radial glia. It remains unknown whether radial glial cells or their precursors, the radial cells, or both can give rise to neurons in the spinal cord. Radial glial cells are also important in regulating the axon out‐growth and pathfinding processes that occur during white matter patterning of the developing spinal cord.

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O2 levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O2 imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O2 in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O2 levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O2 in 3D tissue models.

Differential effects of growth/differentiation factor 5 and glial cell line-derived neurotrophic factor on dopaminergic neurons and astroglia in cultures of embryonic rat midbrain

Parkinsons disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Several studies have examined the effects of the dopaminergic neurotrophins growth/differentiation factor 5 (GDF5) and glial cell line‐derived neurotrophic factor (GDNF) on these neurons in vitro. However, there is little information regarding their effects on astroglial cells. Here, the effects of GDF5 and GDNF on dopaminergic neuronal and astroglial survival and differentiation in embryonic rat midbrain cultures were examined. Both GDF5 and GDNF enhanced the survival and differentiation of dopaminergic neurons. GDF5 significantly increased the survival of astroglial cells, whereas GDNF had no significant effect on these cells. The possible involvement of astroglia in the dopaminergic neurotrophic effect induced by GDF5 was investigated by examining the effect of GDF5 on the survival of dopaminergic neurons in glia‐depleted midbrain cultures. There was no significant difference between the survival of dopaminergic neurons in glia‐depleted cultures treated with GDF5 and that in mixed cell cultures treated with GDF5, suggesting that GDF5 acts directly on dopaminergic neurons in exerting its neurotrophic effect. GDF5 and GDNF have been established as potent neurotrophic factors for dopaminergic neurons. However, the effects of adding a combination of these neurotrophins to midbrain cultures have not been previously examined. The present study found that combined treatment with GDF5 and GDNF significantly increased the survival of dopaminergic neurons in cultures compared with that in cultures treated with either neurotrophin alone. This was an additive effect, indicating that these neurotrophins act on separate subpopulations of dopaminergic neurons.

Radial glial cells: key organisers in CNS development.

Radial glia are elongated bipolar cells present in the CNS during development. Our understanding of the unique roles these cells play has significantly expanded in the last decade. Historically, radial glial cells were primarily thought to provide an architectural framework for neuronal migration. Recent research reveals that radial glia play a more dynamic and integrated role in the development of the brain and spinal cord. They represent a major progenitor pool during early development and can give rise to a small population of multipotent cells in neurogenic niches of the adult CNS. Radial glial cells are a heterogeneous population, with divergent and often poorly understood roles across different brain and spinal cord regions during development; this heterogeneity extends to specialised adult subtypes, such as tanycytes, Müller glial cells and Bergman glial cells which possess morphological similarities to radial glial but play distinct functional roles in the CNS.

Oxygen-sensing scaffolds for 3-dimensional cell and tissue culture

Porous membrane scaffolds are widely used materials for three-dimensional cell cultures and tissue models. Additional functional modification of such scaffolds can significantly extend their use and operational performance. Here we describe hybrid microporous polystyrene-based scaffolds impregnated with a phosphorescent O2-sensitive dye PtTFPP, optimized for live cell fluorescence microscopy and imaging of O2 distribution in cultured cells. Modified scaffolds possess high brightness, convenient spectral characteristics (534 nm excitation, 650 nm emission), stable and robust response to pO2 in phosphorescence intensity and lifetime imaging modes (>twofold response over 21/0% O2), such as confocal PLIM. They are suitable for prolonged use under standard culturing conditions without affecting cell viability, and for multi-parametric imaging analysis of cultured cells and tissue samples. We tested the O2 scaffolds with cultured cancer cells (HCT116), multicellular aggregates (PC12) and rat brain slices and showed that they can inform on tissue oxygenation at different depths and cell densities, changes in respiration activity, viability and responses to drug treatment. Using this method multiplexed with staining of dead cells (CellTox Green) and active mitochondria (TMRM), we demonstrated that decreased O2 (20-24 μM) in scaffold corresponds to highest expression of tyrosine hydroxylase in PC12 cells. Such hypoxia is also beneficial for action of hypoxia-specific anti-cancer drug tirapazamine (TPZ). Thus, O2 scaffolds allow for better control of conditions in 3D tissue cultures, and are useful for a broad range of biomaterials and physiological studies.

Morphology and differentiation of radial glia in the developing rat spinal cord

Important events underlying the proper functioning of the central nervous system (CNS) include the production, assembly, and differentiation of appropriate types and numbers of cells during development. The mechanisms that control these events are difficult to unravel because of displacement of cells from their sites of origin to their permanent locations and because of the diverse cellular composition of the CNS. As in other regions of the mammalian CNS, the two major classes of neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. In the developing spinal cord, radial glia are prominent. In this study, radial glia in the cervical region of the spinal cord were analysed. 1,1′Dioctadecyl‐3,3,3′‐tetramethylindocarbocyanine perchlorate (DiI) was used to determine the morphology and distribution of radial glia during spinal cord development. The DiI labelling technique enabled locating glial precursor cells during spinal cord development. Radial fibres that extended from the central canal to the pial surface were present at embryonic days 14, 16, and 18 in the developing spinal cord. Their distribution was restricted with increasing development, and by embryonic day 20 the only remaining evidence of radial glia were short radial processes in the white matter. J. Comp. Neurol. 454:263–271, 2002.

Differentiation of oligodendrocytes in neurospheres derived from embryonic rat brain using growth and differentiation factors

Studies on the isolation and propagation of multipotent neural precursors as neurospheres suggest their potential use in the reconstitution of neurons and oligodendrocytes in neurodegenerative diseases. To ensure that an adequate number of functionally relevant cells are present after transplantation, in vitro manipulation of cell fate before transplantation may be necessary to control the terminal phenotype of these cells. Using growth factors known to have a role in oligodendrocyte development such as sonic hedgehog, platelet‐derived growth factor (PDGF), and basic fibroblast growth factor (FGF‐2), we have tried to increase the number of oligodendroglia derived from E18 cortical neurospheres. We have shown that although all of the growth factor combinations induce the formation of oligodendroglia, they do so in varying proportions, with PDGF favouring the formation of oligodendrocyte progenitor cells and sonic hedgehog favouring the formation of mature oligodendrocytes. To further enhance the generation of oligodendroglia we exposed neurospheres to B104‐cell conditioned medium (B104 CM). Long‐term growth of the neurospheres in this B104 CM increased markedly the number of cells committed to the oligodendrocyte lineage, specifically oligodendrocyte progenitor cells. These were then referred to as oligospheres. Our results suggest that the oligosphere culture system may provide a valuable source of cells for the reconstitution of oligodendrocytes in neurologic disorders.