Antonia Vernadakis

GLIA-NEURON INTERCOMMUNICATIONS AND SYNAPTIC PLASTICITY

It is established that astrocytes are the intimate partner of neurons throughout their lifespan. However, astrocytes play different roles at different stages of the lifespan. During neurogenesis and early development, glial cells provide a scaffold for the correct migration of neurons and growth cones. They provide guidance cues and may also be involved in neuronal proliferation. In the adult, astrocytes maintain neuronal homeostasis and synaptic plasticity. This review discusses some of the cell-cell communication signals which are involved in the maintenance of synaptic plasticity. They are divided into: (a) glia-to-glia signaling, which involves non-synaptic communication by coupling of astrocytes. In this cell communication, cytoplasmic exchange of ions and small molecules among cells is accomplished through cell coupling of cells via cell-to-cell contacts, termed gap junctions; (b) neuron-to-glia signaling, which involves synaptic interactions. This cell-cell intercommunication has received considerable attention. Of special interest is the role of astrocytes in glutamic acid compartmentation and in preventing glutamic acid neurotoxicity. A glutamate-induced calcium signaling appears to be involved in this neuron-astrocyte interaction. The intriguing possibility is that neuronally induced astrocyte calcium signals may feed back to influence neuronal excitability or regulate synaptic transmission; (c) signals from astrocytes to neurons: a new concept of neuron-glia intercommunication. As mentioned, calcium appears to be the molecule in this glia-neuron signaling, although glial cell receptor-mediated signals are involved also. Receptor interactions on astrocytes through a cascade of events lead to modification in the extracellular concentration of glutamate. The role of astrocytes in synaptic plasticity is not as well understood during ageing and their role in neuronal cell death during ageing and neurodegeneration can only be speculated. However, astrocytes in the aged brain exhibit various receptors, including glutamate receptors. Thus, astrocytes can be expected to modify the expression of endogenous neurotoxins and thus contribute to synaptic plasticity in ageing. Synaptic plasticity continues to be a homeostatic relationship between neurons and glial cells. The possibility of signaling from astrocytes to neurons has opened new horizons for glial cell function and new challenges of research for gliobiologists.

Neuron-Glia Interrelations

Considerable progress in our understanding of neuron and glial cell interrelationships has emerged during the last decade from in vitro and in vivo studies. Neural culture systems have provided powerful tools to delineate cellular and molecular events. Moreover, the advances in development of immunocytochemical and biochemical specific cell markers has made possible the characterization of complex cell behaviors. Glial cells actively participate in several aspects of neuronal growth and differentiation both by providing cell-cell contact interactions and by secreting neuronal growth-promoting factors. In turn, neurons influence the cellular behavior of both astrocytes and oligodendrocytes, primarily by secreting substances into the microenvironment. Such substances as neurohormones and neurotransmitters have been shown to affect several glial functions including electrophysiological responses, energy metabolism, and ionic homeostasis. In several instances these effects appear to be mediated through receptors on glial cells. Astrocytes actively participate in the regulation of the ionic environment. They take up and release several neurotransmitter substances and can modulate the concentration of a neurotransmitter substance at the synaptic cleft and thus monitor neuronal activity. The evidence of neuron-astroglia synaptic contacts supports the view that such contacts are present during early neuroembryogenesis and thus may provide contact signals for neuronal growth. The process of myelination in the CNS appears to be regulated by both neuronal signals to the oligodendrocyte and also intrinsic programming in the oligodendrocytes to produce myelin components. The prevailing view that astrocytes impede regeneration appears to be shifting towards a more favorable notion of the role of these cells in promoting this process. Of interest is the concept that there is a critical period in the ability of astrocytes either to enhance regeneration or to form a gliotic scar and impede this process. The role of glial cells in the aging process of the neuron is only beginning to be appreciated. If glial cells are actively involved in the regulation of the microenvironment, then it follows that any changes in the behavior of glial cells with aging will ultimately affect neuronal function. It is abundantly clear from in vitro studies that glial cells are pluripotential cells with several functional capabilities. Their responsiveness to an environment in which neurons are maturing as compared to an environment where neurons are injured or aging clearly portrays the multifunctional role of the astrocyte.(ABSTRACT TRUNCATED AT 400 WORDS)

The Developing Animal as a Model

Evidence is presented that some excitatory systems are mature at birth and that inhibitory systems develop rapidly during the first 3 weeks after birth.

Morphological and biochemical study in X-ray- and dibutyryl cyclic AMP-induced differentiated neuroblastoma cells☆

Abstract X-irradiation or dibutyryl adenosine 3′:5′-cyclic monophosphate (DBcAMP) induced axon formation in mouse neuroblastoma cells in vitro. The differentiated cells showed morphological maturation, as shown by an increase in the cellular and nuclear size. Neuroblastoma cells in the presence of DBcAMP grew at a slightly reduced rate for 2 days; however, at day 3 they reached plateau phase. The removal of DBcAMP and readdition of fresh growth medium at day 4 did not cause renewal of cell division, indicating that morphological cell differentiation for the most part was irreversible. Na-butyrate, 3′5′ cyclic AMP, 5′ AMP, ATP, ADP, 3′5′ cyclic guanosine monophosphate caused inhibition of cell growth but did not produce morphological differentiation. Acetylcholinesterase activity in both the X-ray- and DBcAMP-induced differentiated cells increased by a factor of 10. Na-butyrate and 3′5′ cyclic AMP which inhibited cell division but did not cause morphological differentiation also increased AChE activity to a similar extent.

Early and late passage C-6 glial cell growth: similarities with primary glial cells in culture.

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into “astrocytic phenotype”.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.

Effects of ethanol on cultured glial cells: proliferation and glutamine synthetase activity ☆

The development of glial-enriched cultures prepared from 15-day-old embryonic chick cerebral hemispheres was examined after exposure to ethanol. Between culture days 6 and 10, ethanol was added to the medium at 4 concentrations: 0.1%, 0.5%, 1.0% and 2.0% (w/v). During the drug exposure interval, morphologic maturation of the cultures was surveyed with phase microscopy; cell proliferation was assessed by cell counts and DNA content. Exposure to ethanol concentrations of 0.1% and 0.5% resulted in only minimal changes in the parameters investigated, whereas biosynthetic and enzymatic activities were altered at a threshold dose of 1.0% ethanol. A small number of cells resembling reactive astrocytes were encountered in cultures exposed to 1.0% and 2.0% ethanol. In these cultures the number of cells and DNA content were lower than in control cultures. Additionally, [14C]leucine incorporation into protein and glutamine synthetase activity were markedly diminished in cultures treated with 1.0% and 2.0% ethanol. The ethanol-related reduction in glutamine synthetase activity was not accompanied by an equivalent decrease in protein content, suggesting that glutamine synthetase or cells rich in this enzyme are selectively vulnerable to ethanol. Impairment in glutamate/glutamine metabolism may be a potential consequence of ethanol-induced changes in glutamine synthetase activity.

Characterization and transplantation of two neuronal cell lines with dopaminergic properties

Immortalized rat mesencephalic cells (1RB3AN27) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP2) in culture. Treatment of 1RB3AN27 and NBP2 cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB3AN27 cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB3AN27 cells exhibited β- and α-adrenoreceptors, and prostaglandin E1 receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D1) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKCα and PKCβ isoforms were higher than those of PKCγ and PKCδ in 1RB3AN27 cells. The 1RB3AN27 cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP2 cells. The grafted 1RB3AN27 were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.

SummaryThis investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400µg/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV3neo, revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.

Urinary cortisol excretion rates and anxiety in normal 1-year-old infants.

&NA; Urinary cortisol excretion rates were determined for each voiding during 8 hours on 3 days for 20 infants. The first day served as the control. On the second day stress was imposed by having the mother leave the infant for an hour. On a third day the child was stimulated by novel toys and socialization for an hour in the mothers presence. There was no significant difference between mean cortisol excretion rates on control and stress days. Variability in cortisol levels was significantly greater on the day of stress than on the other 2 days. Ratings of separation anxiety and levels of cortisol excreted 40 to 160 min after the imposed stress were positively correlated (r = 0.46, P < 0.01). Cortisol levels also differentiated between anxious infants who were agitated from those who were withdrawn. No altered levels of cortisol were associated with excitement.

Growth factors attenuate the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo

The interaction between growth factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase activity as a cholinergic marker. As we have previously reported (Brodie & Vernadakis, Dev. Brain Res.56: 223–228, 1990; Kentroti and Vernadakis, Dev. Brain Res.56: 205–210, 1990), ethanol administration in ovo at embryonic days 1–3 produced a 30% decrease in choline acetyltransferase activity. Nerve growth factor and epidermal growth factor administration alone, at embryonic days 1–3, produced a slight increase in choline acetyltransferase activity of both brain and spinal cord when examined at embryonic day 8. Concomitant administration of either nerve growth factor or epidermal growth factor with ethanol eliminated the decrease in choline acetyltransferase activity produced by ethanol. Moreover, administration of either nerve growth factor or epidermal growth factor at embryonic days 4–7 to embryos pretreated with ethanol at days 1–3 raised choline acetyltransferase activity to a level similar to that observed in controls. Thus the growth factors reversed the ethanol‐induced cholinergic insult and restored the cholinergic population to normal.