Synapses between NG2 glia and neurons

NG2-expressing glia are precursors to oligodendrocytes and subpopulations of astrocytes. They are unique among glial cells in that they enter into synaptic specialisations with neurons throughout all areas of grey and white matter and at all ages. To date, the NG2 cells appear to represent a postsynaptic compartment, and synapses are formed with axons. With differentiation to oligodendrocytes, NG2 is downregulated and myelin antigens upregulated: this coincides with a loss of the synaptic contacts between neurons and NG2 glial cells. The functional roles of this glial-neuron synapse in regulation of differentiation into myelinating oligodendrocytes or additionally responding to and modulating neuronal network activity remain to be elucidated.     

On the Ca2+-permeability of neurons and glia

Fractions enriched in pinched-off nerve terminals and astrocytic glial cells were used to analyse the permeability of Ca⁺⁺of the two membranes. Three experimental models were used to illustrate a principle difference of the excitatory versus the non-excitatory cell membrane, with respect to Ca-permeability. The effect of an increased [Ca⁺⁺] on ⁸⁶Rb⁺and ³H-GABA transport was measured in medium containing low and high [K⁺]. The possibility of GABA stimulation of release of preaccumulated ⁴⁵Ca⁺⁺with low and high [K⁺] was compared in neurons and glia. Finally, the effect of depolarizing [K⁺] on the transmembranal Ca⁺⁺-gradient was measured by comparing the efficacy of the Ca-ionophore A23187 to depolarize or to inhibit the ³H-GABA uptake at these different [K⁺]. The data essentially confirms the picture of a potential dependent Ca⁺⁺-permeability in the neuron, while the permeability of the glial cells seems a “true” constant. It might be relevant to suggest the astrocyte a role as a “Ca-buffer” with possibilities to control extracellular Ca⁺⁺in cases of hyperactivity, this in analogy with what has been suggested for K⁺.     

Differentiation of the human NT2 cells into neurons and glia

A method underlying a strategy for differentiation of the NT2 human teratocarcinoma cell line into neuronal and glial cells is described. The aim of this work is to provide a human model to study the relationships between neurons and glia in vitro during developmental or degenerative events. NT2 cells are seeded on polylysine precoated plastic or glass and differentiated by all-trans retinoic acid; persistent undifferentiated cells are eliminated by cytosine--D-arabinofuranoside; then cell cultures are maintained during four weeks until the appearance of glutamatergic receptors. Along the differentiation procedure, we have followed the expression of neuronal and glial phenotypes as well as the excitotoxic response to N-methyl-D-aspartate treatment taken as an indication of neuronal maturation. The procedure described leads to the development of a mixed population of neurons and glia sensitive to glutamate exposure.     

Moving background patterns reveal double-opponency of directionally specific pigeon tectal neurons

Summary The experiments reported in this paper were carried out to determine the effect moving background patterns have on the response characteristics of directionally specific neurons in the pigeon optic tectum. First, care was taken to select the optimal single stimulus for each cell, then large textured patterns were added to the test stimulus and moved either in-phase or anti-phase. Altogether 214 cells were studied in 77 white Carneaux pigeons and it was found that all cells below a depth of 400 microns were inhibited by backgrounds moved in-phase with the optimal test stimulus, while few cells above this level were affected in any way by backgrounds. All directions of background motion containing an in-phase vector resulted in rather profound inhibition of the directional response while directions with an anti-phase vector produced less inhibition and sometimes were even facilitated by direct anti-phase. The velocity tuning curves obtained with an optimal single test stimulus and by anti-phase movement of backgrounds were essentially similar.In-phase inhibition can also be produced by a second spot stimulus located some distance from the test stimulus. This latter effect was used to map the outer margins of the inhibitory receptive fields of deep tectal neurons displaying these effects and it was found they were extremely large, often in excess of 100 ° in diameter. When masks were used to prevent the moving background from stimulating the excitatory receptive field, anti-phase movement always produced facilitation. This suggests a double opponent-process directionally specific receptive field organization.These neurons seem well suited to respond to local (object) motion and to ignore translation of the visual image arising from body, head and eye movements.Supported by a grant No. A0353 from the Natural Sciences and Engineering Research Council of Canada to B.J. Frost     

Epidermal growth factor affects both glia and cholinergic neurons in septal cell cultures.

The effects of epidermal growth factor on high density primary cultures of fetal (embryonic day 17) rat septal cells were examined. Under serum-free conditions, the continuous exposure of these cultures to epidermal growth factor for seven days significantly decreased choline acetyltransferase (EC 2.3.1.6) activity in a dose-dependent manner. Maximal decreases were observed from 1 to 10 ng/ml epidermal growth factor. This effect was completely abolished by the addition of anti-epidermal growth factor antibodies. The epidermal growth factor-mediated decrease in choline acetyltransferase activity was culture-time dependent, being first detectable after five days of factor application and may likely represent an inhibition of the spontaneous increase in enzyme activity that occurs with time in culture. Concomitant with changes in enzyme activity, epidermal growth factor produced a significant and proportional decrease in the number of acetylcholinesterase-positive neurons. This decrease in acetylcholinesterase-positive cells did not reflect a decrease in cholinergic cell survival as nerve growth factor could restore the number of acetylcholinesterase-positive neurons in epidermal growth factor-treated cultures to control levels. Furthermore, in these high-density cultures, epidermal growth factor did not affect general neuronal survival, while it did produce an increase in the number and intensity of glial fibrillary acidic protein-immunoreactive astroglia as well as in the number of macrophage-like cells. The proliferative response of these non-neuronal cells to epidermal growth factor, as assessed by [3H]thymidine incorporation, was evident after three days of epidermal growth factor application, persisted thereafter, and could be antagonized by the inclusion of the antimitotic 5-fluorodeoxyuridine. Furthermore, 5-fluorodeoxyuridine completely blocked the epidermal growth factor-mediated decrease in choline acetyltransferase activity. However, when epidermal growth factor was tested in pure glial cultures, it only directly induced proliferation of astrocytes. These results suggest that the proliferative response of either one or both of these glial cell types in the mixed cultures may be indirectly affecting cholinergic cell expression.     

Efflux of choline from neurons and glia in culture

The efflux of radioactive choline from exclusively neuronal or glial cell cultures was dependent upon the concentrations of choline present in the cells and in the incubation medium, suggesting the possible presence of a homoexchange phenomenon between influx and efflux. The ionic dependence of the outward movement of choline from these cells showed that it could be stimulated by high K⁺ concentrations and by the absence of Ca²⁺. In glial cells, however, the efflux of choline was increased with a much lower concentration of K⁺ compared to neurons. The result may suggest that during nerve stimulation the release of K⁺ from neurons could stimulate, from glia, the efflux of choline which would then be taken up in neurons.     

Subsets of retinal neurons and glia express P2Y1 receptors

Recent evidence suggests that extracellular ATP modulates retinal processing and could play a role in modulating glial cells during retinal diseases. Here, we evaluated the localization of P2Y₁ receptors in the rat retina using indirect immunofluorescence immunocytochemistry. We observed labeling within defined populations of inner retinal neurons and Müller cell processes and end feet. Double labeling of P2Y₁ receptor with choline acetyltransferase revealed extensive colocalization indicating the expression of this receptor by cholinergic amacrine cells. Ganglion cell labeling for P2Y₁ receptors was also observed. Having established the normal pattern of immunolabeling within the retina, we next examined whether immunolabeling was altered by retinal disease. P2Y₁ receptor immunolabeling of Müller cells was of greater intensity following light-induced retinal degeneration, suggesting that Müller cell gliosis is accompanied by changes in P2Y₁ receptor expression. Overall, these data provide further evidence for a role of extracellular ATP in retinal signaling within subsets of retinal neurons as well as glia.     

PDGFRA/NG2 glia generate myelinating oligodendrocytes and piriform projection neurons in adult mice

Platelet-derived growth factor alpha receptor (PDGFRA)/NG2-expressing glia are distributed throughout the adult CNS. They are descended from oligodendrocyte precursors (OLPs) in the perinatal CNS, but it is not clear whether they continue to generate myelinating oligodendrocytes or other differentiated cells during normal adult life. We followed the fates of adult OLPs in Pdgfra-creER(T2)/Rosa26-YFP double-transgenic mice and found that they generated many myelinating oligodendrocytes during adulthood; >20% of all oligodendrocytes in the adult mouse corpus callosum were generated after 7 weeks of age, raising questions about the function of the late-myelinating axons. OLPs also produced some myelinating cells in the cortex, but the majority of adult-born cortical cells did not appear to myelinate. We found no evidence for astrocyte production in gray or white matter. However, small numbers of projection neurons were generated in the forebrain, especially in the piriform cortex, which is the main target of the olfactory bulb.     

Radial glia is a progenitor of neocortical neurons in the developing cerebral cortex

Neocortical neurons are produced by cell division of neural stem cells in the ventricular zone of the cerebral cortex. We investigated the production of neurons by infecting neuroepithelial cells with a modified GFP-recombinant adenovirus. The adenovirus DNA is inherited by only one daughter cell at each cell division and travels one way from the progenitor to the progeny. Since the ventricular zone (VZ) of the embryo neocortex expressed an adenovirus receptor, CAR ubiquitously, morphology and cell-lineage of cells in the VZ could be revealed by the adenovirus infection. Radial glias, cells with a bipolar shape, and spherical cells were found as modified-GFP-positive (mGFP+) in the VZ. The bipolar cells (radial cells) had a radial process not in contact with the pia mater and a growth-cone-like structure at the edge of their radial process, while the radial glias had a process spanning all the cortical layers. Ten hours after viral infection, most mGFP+ cells were radial cells. In the following 8 h, the percentage of mGFP+ radial glias in mGFP+ neocortical cells increased from 18 to 50%, while that in radial/spherical cells decreased from 75 to 19%. The radial glias often divided asymmetrically and produced spherical cells and neuronal precursors. The spherical cells seemed to become radial cells by extending a radial process. The spherical cells, radial cells and radial glias seemed to constitute a proliferating cell cycle during which postmitotic neuronal precursors are produced. The neuronal precursors that inherited the radial processes migrated radially and developed into neocortical neurons. Four days after the viral infection, 97% of mGFP+ cells were neocortical neurons. Here, we propose that the radial glia is a progenitor of neocortical neurons, and that a significant number of radially migrating neurons is guided by their own radial processes connected to the pia mater.     

Real-time thickness measurement of biological tissues using a microfabricated magnetically-driven lens actuator

A fiber optic confocal catheter with a micro scanning lens was developed for real-time and non-contact thickness measurement of biological tissue. The catheter has an outer diameter and rigid length of 4.75 mm and 30 mm respectively and is suitable for endoscopic applications. The catheter incorporates a lens actuator that is fabricated using microelectromechanical systems (MEMS) technology. The lens is mounted on a folded flexure made of nickel and is actuated by magnetic field. Thickness measurements are performed by positioning the catheter in front of the tissue and actuating the lens scanner in the out-of-plane direction. A single-mode optical fiber (SMF) is used to deliver a 785 nm laser beam to the tissue and relay back the reflected light from the tissue to a photomultiplier tube (PMT). When the focal point of the scanning lens passes tissue boundaries, intensity peaks are detected in the reflecting signal. Tissue thickness is calculated using its index of refraction and the lens displacement between intensity peaks. The utility of the confocal catheter was demonstrated by measuring the cornea and skin thicknesses of a mouse. Measurement uncertainty of 8.86 μm within 95% confidence interval has been achieved.     

Influence of a moving textured background on direction selectivity of cat striate neurons

The influence of a moving textured background on direction selectivity for a moving bar was tested in 118 striate neurons and in 19 dorsal lateral geniculate neurons of anesthetized and paralyzed cats. In the standard conditions the background was a two-dimensional noise pattern, the bar moved at optimal speed, and its contrast was adjusted to the level producing 50% of the maximum response. These experiments revealed a new typology of cortical cells based on relative direction selectivity. Six different relative-direction-selectivity types are described. Two types of cells were found to have opposite kinds of relative direction selectivity: antiphase direction-selective cells (5% of the cortical sample) preferred the direction of the bar opposite to the direction of background motion, and absolutely direction-selective cells (20% of the cortical sample) kept their direction selectivity for bar motion independently of the background motion. Three types of cortical cells were direction selective for bar motion only in restricted background motion conditions: conditionally direction-selective cells (20% of cortical sample) only expressed their direction selectivity when the bar and the background moved in antiphase, differencing direction-selective cells (5% of the cortical sample) only expressed their direction selectivity when the bar and the background differed in speed, and limited direction-selective cells (20% of the cortical sample) only expressed their direction selectivity for near zero background speeds. The sixth type, relative nondirection-selective cells (30% of the cortical sample and all of the geniculate cells) were direction selective for none of the background motion conditions. These different relative-direction-selectivity types differed in RF organization, in ocular dominance, velocity sensitivity, in laminar distribution, and in distribution in the visual field. The relative-direction-selectivity types were invariant for changes in the contrast and bar speed. The construction of these relative-direction-selectivity types from the geniculate input requires some inhibitory, but mainly facilitatory, intracortical interactions. These experimental findings suggest that area 17 in the cat has the neuronal machinery to extract depth from motion (limited direction-selective cells) and to segregate visual scenes by motion cues (antiphase, conditionally and differencing direction-selective cells).     

Stimulus-dependent activation of c-Fos in neurons and glia in the rat cerebellum

The intent of the present study was to use chemical or electrical stimulation of cerebellar afferents to determine how different stimulation paradigms affect the pattern of activation of different populations of neurons in the cerebellar cortex. Specifically, we analyzed immediate changes in neuronal activity, identified neurons affected by different stimulation paradigms, and determined the time course over which neuronal activity is altered. In the present study, we used either systemic (harmaline) or electrical stimulation of the inferior cerebellar peduncle (10 and 40 Hz) to alter the firing rate of climbing and mossy fiber afferents to the rat cerebellum and an antibody made against the proto-oncogene, c-fos, as a marker to identify activated neurons and glia. In control animals, only a few scattered granule cells express nuclear Fos-like immunoreactivity. Although no other cells show Fos-like immunoreactivity in their nuclei, Purkinje cells express Fos-like immunoreactivity within their somatic and dendritic cytoplasm in control animals. Within 15 min of chemical or electrical stimulation, numerous granule and glial cells express Fos-like immunoreactivity in their nuclei. Cells in the molecular layer express Fos-like immunoreactivity following harmaline stimulation in a time and lobule specific manner; they do not appear to be activated in the electrical stimulation paradigm. Following harmaline injections, there is an initial loss of Fos-like immunoreactivity in the cytoplasm of Purkinje cells; 90 min later, nuclear staining is observed in a few scattered Purkinje cells. Following electrical stimulation, the cytoplasmic staining in Purkinje cells is enhanced; it is never present in the nucleus. Data derived from this study reveal cell-specific temporal and spatial patterns of c-Fos activation that is unique to each paradigm. Further, it reveals the presence of an activity dependent protein in the cytoplasm of Purkinje cell somata and dendrites.     

Differentiation of postnatal neural stem cells into glia and functional neurons on laminin-coated polymeric substrates

A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp. The results show that the substrate's surface chemistry influences cell attachment and neuronal differentiation, probably through its influence on adsorbed laminin, and that copolymers based on PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and differentiation of adult NSCs into functional neurons and glia.     

Membrane capacitance of cortical neurons and glia during sleep oscillations and spike-wave seizures

Dual intracellular recordings in vivo were used to disclose relationships between cortical neurons and glia during spontaneous slow (<1 Hz) sleep oscillations and spike-wave (SW) seizures in cat. Glial cells displayed a slow membrane potential oscillation (<1 Hz), in close synchrony with cortical neurons. In glia, each cycle of this oscillation was made of a round depolarizing potential of 1.5-3 mV. The depolarizing slope corresponded to a steady depolarization and sustained synaptic activity in neurons (duration, 0.5-0.8 s). The repolarization of the glial membrane (duration, 0.5-0.8 s) coincided with neuronal hyperpolarization, associated with disfacilitation, and suppressed synaptic activity in cortical networks. SW seizures in glial cells displayed phasic events, synchronized with neuronal paroxysmal potentials, superimposed on a plateau of depolarization, that lasted for the duration of the seizure. Measurements of the neuronal membrane capacitance during slow oscillating patterns showed small fluctuations around the resting values in relation to the phases of the slow oscillation. In contrast, the glial capacitance displayed a small-amplitude oscillation of 1-2 Hz, independent of phasic sleep and seizure activity. Additionally, in both cell types, SW seizures were associated with a modulatory, slower oscillation ( approximately 0.2 Hz) and a persistent increase of capacitance, developing in parallel with the progression of the seizure. These capacitance variations were dependent on the severity of the seizure and the distance between the presumed seizure focus and the recording site. We suggest that the capacitance variations may reflect changes in the membrane surface area (swelling) and/or of the interglial communication via gap junctions, which may affect the synchronization and propagation of paroxysmal activities.     

Orderly arrangement of hepatocyte spheroids on a microfabricated chip

This article describes a novel method for preparing several spherical multicellular aggregates (spheroids) that have almost the same diameter on a microfabricated chip. The chip, fabricated by a simple method that uses a micromilling system, consisted of several thousand cavities, 100-500 microm in diameter, in a triangular arrangement on a polystyrene plate. Although no spheroid was formed on any chip when cultured under stationary conditions, hepatocytes formed spheroids in the cavities when turning force was applied with a rotary shaker. Especially on the chip with cavities 300 microm in diameter, one spheroid formed in each cavity; an orderly array of spheroids (1100 spheroids/cm(2)) of almost the same diameter was constructed. Ammonia removal and albumin secretion by the spheroids on the chip continued to occur at initial levels for at least 14 days of culture. Enzyme P-450 activity of the spheroids was also maintained and detected on this transparent chip by fluorescence of resorufin converted from ethoxyresorufin. The spheroid microarray chip seems to be a promising cellular platform for various biomedical applications such as in cell-based biosensors for toxicological and pharmacological examinations, and in bioartificial livers.     

Immunohistochemical investigation of protein S-100 in neurons and glia of Helix pomatia

The location of brain-specific protein S-100 was investigated by the indirect Coons' method in neurons and glia ofHelix pomatia. This protein was found in the cytoplasm of neurons and glial cells, and in the nucleus and outer member of neurons.The antiserum was provided by Candidate of Biological Sciences S. M. Sviridov of the Laboratory of Genetic Bases of Ontogeny (Head, Professor L. I. Korochkin), Institute of Cytology and Genetics, Siberian Branch, Academy of Sciences of the USSR, to whom the writers are grateful.Laboratory of Neurophysiological Mechanisms of Adaptation, Institute of Clinical and Experimental Medicine, Academy of Medical Sciences of the USSR, Siberian Branch, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 12, pp. 1501–1503, December, 1976.     

Control of cellular organization in three dimensions using a microfabricated polydimethylsiloxane-collagen composite tissue scaffold

Parallel channels of various dimensions have been shown to cause a monolayer of cells in culture to align in the direction of the channels. For the engineering of complex organ systems to become a reality, similar control over the cellular microenvironment in three dimensions must be achieved. Using microfabrication, a polydimethylsiloxane (PDMS) scaffold (40 microm wide, 70-microm-deep parallel channels separated by 25-microm-wide walls) was created. A fibroblast-seeded collagen matrix was then molded around this PDMS scaffold. The PDMS scaffold served as an internal skeleton to guide the cells to grow in the prescribed three-dimensional pattern. Organization, aspect ratio, and the z diameter of the cells were analyzed by confocal microscopy. Fibroblasts elongated and organized in the direction of the channels throughout the height of the scaffold. The mean angle of the cells off of the long axis of the channels was 4.3 +/- 0.7 degrees as opposed to 32.6 +/- 2.2 degrees in controls. The morphology of the cells was also affected by the PDMS scaffold. The nuclei were longer (1.25x) and thinner (0.75x) than in control gels; however, no changes in diameter of the cells in the z direction were seen.