Charles F. Ide

Temporal characterization of microglia, II-1β-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures

These studies demonstrate that murine hippocampal slice cultures possess neural‐immune elements that show responses parallel to comparable in vivo models of neural‐immune activation. Using immunocytochemical techniques, this study characterized the phenotypes of specific glial elements and the expression of the cytokine, interleukin‐1 (IL‐1β), in the hippocampal dentate gyrus over a period of 10 days in vitro (DIV). Preparation of organotypic slice cultures of neonatal mouse hippocampus produced cellular damage including axotomy of afferent fibers within the molecular layer of the dentate gyrus. This form of lesion‐induced injury caused activation of neural‐immune elements in the slice cultures. Staining with the microglial specific, biotinylated Griffonia simplicifolia B4‐isolectin revealed reactive microglia were most prevalent at 2 DIV and decreased in number from 4 to 10 DIV, whereas the initial population of resting microglia at 2 DIV increased approximately four‐fold from 4 to 10 DIV. The presence of a round IL‐1β‐like immunophenotype closely paralleled the temporal and spatial distribution of the reactive form of microglia observed in the dentate gyrus. In addition, between 4 and 10 DIV, some IL‐1β‐like immunoreactive cells exhibited a stellate‐like morphology with numerous branching processes, similar to resting microglia. At 2 DIV, astrocytes showed minimal labeling with antibodies directed against glial fibrillary acidic protein (GFAP), while from 4 to 10 DIV, a dramatic hypertrophic astrocytic response occurred, resulting in a gliotic scar forming over the entire dentate gyrus. We conclude that neural‐immune activation in the hippocampal organotypic slice culture preparation closely parallels similar responses observed in vivo and thus slice cultures represent an excellent model for further studies of neural‐immune interactions resulting from lesion‐induced injury in the central nervous system.

Aging in the hippocampus: Interrelated actions of neurotrophins and glucocorticoids

Over the past two decades, evidence has been accumulating that diffusible molecules, such as growth factors and steroids hormones, play an important part in neural senescence, particularly in the hippocampus. There is also evidence that these molecules do not act as independent signals, but show interrelated regulation and cooperative control over the aging process. Here, we review some of the changes that occur in the hippocampus with age, and the influence of two classes of signaling substances: glucocorticoids and neurotrophins. We also examine the interactions between these substances and how this could influence the aging process.

Interleukin-1β and its type 1 receptor are expressed in developing neural circuits in the frog, Xenopus laevis

The cytokine interleukin‐1 beta (IL‐1β) is an evolutionarily conserved molecule that was originally identified in the immune system. In addition to regulating peripheral immune responses, IL‐1β plays an important role in mediating neural‐immune interactions and regulating glial activities during healing and repair in the damaged nervous system. Active IL‐1β is produced by interleukin‐converting enzyme (ICE), a caspase thought to be involved in the induction of apoptosis. We report that, in the developing frog, Xenopus laevis, IL‐1β and the IL‐1 type 1 receptor proteins are coexpressed in specific neurons that comprise early sensory‐motor circuits. IL‐1β and IL‐1 type 1 receptor proteins are colocalized in specific midbrain and hindbrain reticular cells, including Mauthners neuron; specific cells in the trigeminal (fifth), lateral line (seventh), and vestibular (eighth) cranial ganglia; oculomotor neurons; and the primordial Purkinje cells of the lateral cerebellar auricle. In the spinal cord, Rohon‐Beard sensory neurons, dorsal root ganglion cells, and primary motoneurons are immunopositive. Anteriorly, the olfactory pits, olfactory nerves, and olfactory bulbs are labeled, as are retinal cells, especially photoreceptor inner segments. With regard to the function of IL‐1β during neural development, IL‐1β and its type 1 receptor are present throughout the course of neural development in identifiable, long‐lived neurons, such as Mauthners neuron. These and other data suggest that IL‐1β and its type 1 receptor may be involved in the maintenance of cell survival rather than induction of neuronal death. J. Comp. Neurol. 394:242–251, 1998.

Regulation by Interleukin-1β of Formation of a Line of Delimiting Astrocytes Following Prenatal Trauma to the Brain of the Mouse

The regulation of perinatal glia limitans (GL) reformation by interleukin-1beta (IL-1beta) following prenatal neural trauma in the mouse was studied in lesioned fetal mice by immunocytochemistry and computer-assisted image analysis for presence and distribution of astrocytes and IL-1beta immunoreactivity (ir). Astrocytes stained with anti-glial fibrillary acidic protein (GFAP) were observed as a line of delimiting astrocytes (LDA) near the lesion edge on Postnatal Day 0 (P0, 2 days postlesion). At P6, a new and complete GL composed of GFAP-positive astrocytes was continuous with that of adjacent undamaged tissue. The new GL was located in the same area at P6 as was the LDA at P0, suggesting that the LDA is the precursor structure to a reformed GL. Astrocytes comprising the new GL were positive for anti-IL-1beta. The IL-1 receptor antagonist (IL-1ra), administered acutely into the lesion, produced a significantly decreased optical density of IL-1beta-ir at the LDA at P0 compared to animals that received injections of vehicle, human recombinant IL-1beta, or a combination injection of IL-1ra + IL-1beta. Furthermore, although GFAP-stained cells appeared at the lesion site, an organized LDA was not visible at P0 in IL-1ra-treated animals. Vehicle-, IL-1beta-, and combination-injected animals showed a robust LDA at the lesion site at P0. These data suggest that upregulation of IL-1beta in astrocytes and interaction of IL-1beta with the neural IL-1 receptor are important for reconstruction of the GL following prenatal lesion in the murine brain.

Electromagnetic field induced changes in lipid second messengers

Initial studies with a human hematopoietic cell line, TF-1, suggest multifarious effects of electromagnetic fields on lipid signal transduction. We have examined the effects of pulsed magnetic fields (2 T, 84 microseconds zero-to-peak haversine, 91 V/m induced electric field) on the cell cycle by flow cytometry. A 31% increase of cells in the G1 phase occurred concurrently with a 35% decrease of cells in S-phase, which suggests that doses of 30 or 40 pulses have an anti-proliferative effect. Changes in the lipid second messengers, diacylglycerol (DAG) and phosphatidic acid (PA) with stimuli of 2 T intensity were also dependent on the number of pulses. DAG production doubled with 30 pulses and tripled with 40 pulses, and PA levels were reduced to one third and one tenth of the original levels. Phospholipase D (PLD) up-regulation was assessed directly by the capacity of PLD to catalyze transphosphatidylation in the presence of alcohol. [3H]Phosphatidylethanol formed rapidly and continued to increase with concomitant decreases in [3H]PA and parallel generation of [3H]DAG. Propranolol, an inhibitor of PA phosphohydrolase, inhibited the formation of DAG in a dose-dependent manner with a marked increase in PA production. Examination of the kinetics of formation of [3H]choline and [3H]phosphocholine at different times after stimulation showed a rapid and consistent increase in [3H]choline, whereas [3H]phosphocholine increase was evident only 60 min after stimulation. Magnetic exposure also caused a shift in some molecular species patterns of DAG and PA which could be correlated with phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine molecular species decreases. Therefore, we propose that the PC-PLC pathway may be temporarily inactivated for a short period of time by exposure to pulsed stimuli, and the PC-PLD pathway is up-regulated based on: (1) cellular release of [3H]choline; (2) rapid intracellular formation of [3H]PA followed by [3H]DAG; (3)active transphosphatidylation; and (4) blockade of DAG formation by propranolol.

Effects of Alterations of Cell Size and Number on the Structure and Function of the Xenopus Laevis Nervous System

The evolution of the vertebrate central nervous system has involved increasing cell numbers and overall relative size, allometric changes in different brain regions, and quantitative and qualitative alterations in the interactions between different regions of the nervous system. The importance of genetic changes which alter the metrics of interactions between developing organ systems for evolutionary change has been stressed by Gould (1977). Yet relatively little evidence is available from experimental studies to support the idea that allometric changes, such as changes in cell size, cell number or the relative sizes of different organs within the vertebrate nervous system can cause qualitative as well as quantitative changes in structure and function. Recent advances in the genetics of Xenopus laevis have permitted us to investigate some of these effects. The results support the hypothesis that relatively simple alterations in cell size and numbers can cause both qualitative and quantitative changes in morphology and function of the nervous system.

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase.

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G0/G1 phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15–60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.

Survey for magnetically induced plasma membrane damage

An in vitro study using the N1E-115 neuroblastoma cell line was performed to determine whether cell membrane damage occurs during magnetic stimulation. Trypan Blue stain and Fluorescein Isothiocyanate (FITC) dextran uptake was used as an indicator of increased permeability. No acute effects were found.

Duplication of positional information in regeneration of eye-bud fragments results from a specific healing pattern

Fragment cultures of embryonic mouse neural tube were used to study sequential changes in neuronal cell differentiation. We e~amined expression of intermediate filaments (vimentin type, VIF; neurofilaments, NF) and related their patterns to the appearance of other phenotypic markers of neuronal development such as presence of tetanus toxin receptors (TI~, migration of cells, formation of Nissl substance, and the degree of neurite formation. Newly formed neurons, bipolar with scant Nissl substance, were close to the fragment (least migration). Tney were qTR-/NF-/VIF +, with VIF in a perinuclear arrangement e~tending into some processes. Neurons in the middle of the outgrowth zone (average migration) were TTR+/NF+-/VIF~-. Tne morphologically most differentiated neurons, large multipolar cells with abundant Nissl substance, appeared furthest from the fragment (extensive migration) and were TPR+/NF ~rfF-. Between days 7 and i0 of culturing neurons at all stages of differentiation were observable within the outgrowth zone. Neuronal migration appeared limited to the first 10-12 days of culture though neurons continued to differentiate throughout the culture period of 21 days. Tnis system thus provides a model by which mechanisms regulating neuronal proliferation and differentiation in vitro may be studied. Supported by MNC of Canada Grant MT 4235.