Richard M. Devon

Mapping metals in Parkinson's and normal brain using rapid-scanning x-ray fluorescence

Rapid-scanning x-ray fluorescence (RS-XRF) is a synchrotron technology that maps multiple metals in tissues by employing unique hardware and software to increase scanning speed. RS-XRF was validated by mapping and quantifying iron, zinc and copper in brain slices from Parkinsons disease (PD) and unaffected subjects. Regions and structures in the brain were readily identified by their metal complement and each metal had a unique distribution. Many zinc-rich brain regions were low in iron and vice versa. The location and amount of iron in brain regions known to be affected in PD agreed with analyses using other methods. Sample preparation is simple and standard formalin-fixed autopsy slices are suitable. RS-XRF can simultaneously and non-destructively map and quantify multiple metals and holds great promise to reveal metal pathologies associated with PD and other neurodegenerative diseases as well as diseases of metal metabolism.

Olfactory ensheathing cells do not requirel-ascorbic acid in vitro to assemble a basal lamina or to myelinate dorsal root ganglion neurites

Ensheathing cells reside within both the PNS and CNS portions of the primary olfactory pathway and provide a glial covering and support for the unmyelinated olfactory axons. In vivo, these ensheathing cells express a mixture of astrocyte-specific and Schwann cell-specific phenotypic features. When grown in vitro in the presence of DRG neurons however, these ensheathing cells were observed to myelinate DRG neurites. The purpose of the present study was to determine whether ensheathing cells, like Schwann cells, require the addition of ascorbic acid to the medium in order to assemble a basal lamina and a myelin sheath. Our findings indicate that ensheathing cells can myelinate DRG neurites regardless of whether ascorbic acid is included in the growth medium and that these glial cells can assemble a basal lamina in the absence of added ascorbic acid. It appears from these results that Schwann cells and ensheathing cells have different growth media requirements for the assembly of a basal lamina.

Macromolecular translocation — a possible function of astrocytes

We have used dense astrocytic cultures, which display a multilamellar geometry, to determine whether translocation of macromolecules can occur across astrocytic processes. Colloidal gold-labelled transferrin and serum albumin were allowed to bind to the most superficial of the astrocytic cell membranes at 4 degrees C, the temperature was then increased to 37 degrees C and the fate of these gold-labelled macromolecules was observed using the electron microscope. The gold-labelled macromolecules appeared to undergo receptor-mediated endocytosis followed by translocation and exocytosis, with the colloidal gold-labelled macromolecules moving from the apical to the more basal processes. After 1 h of incubation at 37 degrees C, colloidal gold became accumulated within secondary lysosomes of the basal-most layer of astrocyte processes. Since the extracellular space of the central nervous system (CNS) consists of narrow tortuous channels formed to a great extent by the multitude of processes extending from fibrous and protoplasmic astrocytes, these observations suggest to us that one function of astrocytes may be to facilitate transport of macromolecules from one cell to another.

Fibrous astrocytes and reactive astrocyte-like cells in transplants of cultured astrocyte precursor cells

Mouse neopallium was disaggregated at 3 developmental stages (E15, E18, PO) and grown in colony cultures for 7 days. On the seventh day of culturing the colonies of cells were transplanted into the cerebellums of neonatal mice. After 3 weeks the astrocytes within the transplants were identified with GFAP immunoperoxidase staining and by morphometric nuclear measurements of the GFAP positive cells. Cultures of E15 and E18 disaggregated neopallium gave rise to typical fibrous astrocytes in the transplants which were similar to fibrous astrocytes in the cerebral white matter of adult mice. Cultures of PO disaggregated neopallium gave rise to reactive astrocyte-like cells in the transplants which stained intensely for GFAP and had nuclei significantly larger than the astrocytes in the cerebral white matter of adult mice and in transplants of the E15 and E18 cultures.

Comparison of oligodendrocytes grown in neocortex and spinal cord aggregate cultures

Mechanically dissociated cells of mouse central nervous system (CNS) (neocortex and spinal cord) form spherical aggregates in rotation culture and develop into populations of mature neurons and glial cells. Synapses and myelination of axons are evident in these aggregates although onset of these processes differs between aggregate types. In this study neocortex aggregates display synapses at 2 weeks in culture but do not demonstrate myelination of axons until 8 weeks. Spinal cord aggregates demonstrate myelinated axons at 2 weeks in culture although there are few synapses evident. The difference in myelination onset is due in part to the development of predominantly perineuronal oligodendrocytes in neocortex aggregates compared to the development of interfascicular oligodendrocytes in spinal cord aggregates. Both types of oligodendrocytes exhibit light, medium and dark categories and both cell types are capable of myelinating axons in culture.

Muscle derived motoneuron trophic factors promote the survival of motoneurons in vitro only when serum is present in the growth medium

Motoneuron cultures were established from E6 chick spinal cord. Motoneurons survived for less than 2 days in chemically defined medium. The addition of muscle extract to the medium supported the survival of only a small portion (approximately 2%) of motoneurons for 8 days in vitro. A similar low survival rate was observed when the growth medium was supplemented with serum. The addition of muscle extract to serum containing medium resulted in the survival of about 20% of the motoneurons for 8 days. No differences were seen in the ability of tissue extracts prepared from E8 hindlimb, or muscle obtained from E11, E15, E18 and P3 chicks to support motoneuron survival in the presence of serum. It is apparent that although there are trophic factors present in muscle that support motoneuron survival in vitro, the actions of such trophic factors are dependent upon the presence of yet other factors found in serum.

Generation of Oligodendroblasts from Primary Cultures of Rat Neopallium

Several distinct stages of differentiation have been described for the oligodendroglial lineage in vitro (Gard and Pfeiffer, 1990; Gard et al., 1995). These include the actively proliferating bipolar or tripolar oligodendroglial precursor cell, characterized by the presence of GQ1c and GD3 gangliosides in the plasmalemma. The oligodendroglial precursor cell differentiates into the multipolar oligodendroblast, a proliferative cell that has sulfatide but no galactocerebroside in its plasmalemma. The oligodendroblast differentiates into the mitotically quiescent oligodendrocyte, a cell characterized by the presence of galactocerebroside in its plasmalemma. These oligodendrocytes also express other myelin-associated proteins, such as myelin basic protein. When transplanted into the central nervous system (CNS) of hypomyelinating hosts, oligodendrocyte precursors migrate over considerable distances and give rise to large numbers of myelinating oligodendrocytes; oligodendroblasts migrate only short distances and give rise to far fewer myelinating oligodendrocytes (Warrington et al., 1993). When transplanted into the CNS, the mature oligodendrocyte will also myelinate axons (Duncan et al., 1992).