Age-dependent fate and lineage restriction of single NG2 cells

NG2-expressing glia (NG2 cells, polydendrocytes) appear in the embryonic brain, expand perinatally, and persist widely throughout the gray and white matter of the mature central nervous system. We have previously reported that NG2 cells generate oligodendrocytes in both gray and white matter and a subset of protoplasmic astrocytes in the gray matter of the ventral forebrain and spinal cord. To investigate the temporal changes in NG2 cell fate, we generated NG2creER?BAC transgenic mice, in which tamoxifen-inducible Cre is expressed in NG2 cells. Cre induction at embryonic day 16.5, postnatal day (P) 2, P30 and P60 in mice that were double transgenic for NG2creER?BAC and the Cre reporter revealed that NG2 cells in the postnatal brain generate only NG2 cells or oligodendrocytes, whereas NG2 cells in the embryonic brain generate protoplasmic astrocytes in the gray matter of the ventral forebrain in addition to oligodendrocytes and NG2 cells. Analysis of cell clusters from single NG2 cells revealed that more than 80% of the NG2 cells in the P2 brain give rise to clusters consisting exclusively of oligodendrocytes, whereas the majority of the NG2 cells in the P60 brain generate clusters that contain only NG2 cells or a mixture of oligodendrocytes and NG2 cells. Furthermore, live cell imaging of single NG2 cells from early postnatal brain slices revealed that NG2 cells initially divide symmetrically to produce two daughter NG2 cells and that differentiation into oligodendrocytes occurred after 2-3 days.     

Expression of PACAP and glutamate transporter proteins in satellite oligodendrocytes of the human CNS

White matter oligodendrocytes have been shown to actively regulate extracellular glutamate levels in the CNS. Such function has yet not been examined in satellite oligodendrocytes of gray matter. Similar to those in white matter, satellite oligodendrocytes are involved in myelination. In addition, they modulate the activity of surrounding neurons. This study examined whether satellite oligodendrocytes express PACAP and glutamate transporter proteins and whether this expression is influenced by global ischemia. We demonstrated expression of PACAP27 and PACAP38 in a major fraction of satellite oligodendrocytes in normal neocortex and hippocampus of human brain tissues obtained post-mortem. All three glutamate transporters EAAT1, EAAT2 and EAAT3 were expressed in satellite oligodendrocytes from these tissues. Thus, satellite oligodendrocytes may participate in the perineuronal glutamate homeostasis. Following transient global ischemia, the total number of satellite oligodendrocytes expressing PACAP or glutamate transporter proteins was significantly decreased in cerebral neocortex and hippocampus. However, alterations of PACAP and glutamate transporter protein expression were region and time specific. In satellite oligodendrocytes of CA1 an early strong reduction of PACAP and glutamate transporter expression was observed. This contrasted with late reduction of PACAP27, PACAP38 and glutamate transporters EAAT1, EAAT2 and EAAT3 in satellite oligodendrocytes of neocortex. Further studies should clarify whether these alterations in protein expression are primary or secondary to neuronal cell death.     

NG2-expressing cells in the nervous system revealed by the NG2-EYFP-knockin mouse

The NG2 glycoprotein is a type I membrane protein expressed by immature cells in the developing and adult mouse. NG2+ cells of the embryonic and adult brain have been principally viewed as oligodendrocyte precursor cells but have additionally been considered a fourth glial class. They are likely to be a heterogeneous population. In order to facilitate studies on the function of NG2+ cells and to characterize these cells in situ, we generated an enhanced yellow fluorescent protein (EYFP) "knockin mouse." EYFP-expressing cells in heterozygous knockin mice expressed the NG2 protein in all regions and at all ages studied. The EYFP+ cells did not express markers of mature glia, developing or mature neurons or microglia, but expressed markers typical for immature oligodendrocyte-lineage cells. Examination of the hippocampus showed heterogeneity in the population with regard to expression of S100ss and glutamine synthetase. Furthermore, different subpopulations of NG2+ cells in the hippocampus could be recognized by their electrophysiological properties.     

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.     

White matter injury following systemic endotoxemia or asphyxia in the fetal sheep

White matter injury is the most frequently observed brain lesion in preterm infants. The etiology remains unclear, however, both cerebral hypoperfusion and intrauterine infections have been suggested as risk factors. We compared the neuropathological outcome, including the effect on oligodendrocytes, astrocytes, and microglia, following either systemic asphyxia or endotoxemia in fetal sheep at midgestation. Fetal sheep were subjected to either 25 minutes of umbilical cord occlusion or systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (LPS O111:B4, 100 ng/kg, IV). Periventricular white matter lesions were observed in 2 of 6 asphyxiated fetuses, whereas the remaining animals showed diffuse injury throughout the subcortical white matter and neuronal necrosis in subcortical regions, including the striatum and hippocampus. LPS-treatment resulted in focal inflammatory infiltrates and cystic lesions in periventricular white matter in 2 of 5 animals, but with no neuron specific injury. Both experimental paradigms resulted in microglia activation in the white matter, damaged astrocytes, and loss of oligodendrocytes. These results show that the white matter at midgestation is sensitive to injury following both systemic asphyxia and endotoxemia. Asphyxia induced lesions in both white and subcortical grey matter in association with microglia activation, and endotoxemia resulted in selective white matter damage and inflammation.     

NG2 cells generate both oligodendrocytes and gray matter astrocytes

NG2 glia constitute a fourth major glial cell type in the mammalian central nervous system (CNS) that is distinct from other cell types. Although circumstantial evidence suggests that some NG2 glia differentiate into oligodendrocytes, their in vivo fate has not been directly examined. We have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express DsRed or Cre specifically in NG2-expressing (NG2+) cells. In NG2DsRedBAC transgenic mice, DsRed was expressed specifically in NG2+ cells throughout the postnatal CNS. When the differentiation potential of NG2+ cells in vitro was examined using DsRed+NG2+ cells purified from perinatal transgenic brains, the majority of the cells either remained as NG2+ cells or differentiated into oligodendrocytes. In addition, DsRed+NG2+ cells also differentiated into astrocytes. The in vivo fate of NG2 glia was examined in mice that were double transgenic for NG2creBAC and the Cre reporter Z/EG. In the double transgenic mice, the Cre reporter EGFP was detected in myelinating oligodendrocytes and in a subpopulation of protoplasmic astrocytes in the gray matter of ventrolateral forebrain but not in fibrous astrocytes of white matter. These observations suggest that NG2+ cells are precursors of oligodendrocytes and some protoplasmic astrocytes in gray matter.     

Physiological characteristics of NG2-expressing glial cells

Antibodies against the chondroitin sulfate proteoglycan NG2 label a subpopulation of glial cells within the CNS, which have a small cell body and thin radiating processes. Physiological recordings from these small cells in acute brain slices have revealed that they possess unique properties, suggesting that they may comprise a class of glial cells distinct from astrocytes, oligodendrocytes, or microglia. NG2-expressing glial cells (abbreviated as “NG2 cells” here) have a moderate input resistance and are not dye- or tracer-coupled to adjacent cells. They express voltage-gated Na⁺, K⁺and Ca²⁺conductances, though they do not exhibit regenerative Na⁺or Ca²⁺action potentials due to the much larger K⁺conductances present. In addition to voltage-gated conductances, they express receptors for various neurotransmitters. In the hippocampus, AMPA and GABA_Areceptors on these cells are activated by release of transmitter from neurons at defined synaptic junctions that are formed with CA3 pyramidal neurons and GABAergic interneurons. These rapid forms of neuron-glial communication may regulate the proliferation rate of NG2 cells or their development into mature oligodendrocytes. These depolarizing inputs may also trigger the release of neuroactive substances from NG2 cells, providing feedback regulation of signaling at neuronal synapses. Although the presence of Ca²⁺permeable AMPA receptors provides a pathway to link neuronal activity to Ca²⁺dependent processes within the NG2 cells, these receptors also put these cells at risk for glutamate-associated excitotoxicity. This vulnerability to the sustained elevation of glutamate may underlie ischemic induced damage to white matter tracts and contribute to cerebral palsy in premature infants.     

The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells. Implications for glial-neuronal signaling

The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIP1. In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells. GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2.AMPA receptor complex in glial-neuronal recognition and signaling.     

A Brain-Specific Protein p25 Is Localized and Associated with Oligodendrocytes, Neuropil, and Fiber-Like Structures of the CA3 Hippocampal Region in the Rat Brain

Abstract: Developmental expression and cellular localization of a novel brain-specific 25-kDa protein (p25), a substrate of tau protein kinase II, were investigated in the rat brain using polyclonal antibodies raised against peptides synthesized based on the p25 amino acid sequence. By western immunoblotting, p25 was found to be expressed only slightly in the embryonic period; the expression increased from 11 days up to 5 weeks of age, and continued to increase gradually until 1–2 years of age. Immunohistochemistry revealed distinct staining of glial cells in most regions of the central nervous system in the adult rat brain. These positively immunostained cells were especially abundant in the white matter, such as the corpus callosum, cingulum, external capsule, and internal capsule. The glial cells were identified as oligodendrocytes, and the nuclei of the cells remained unstained. Whereas the neuropil in most parts of the brain was immunostained less intensely than glias, the neuropil in the first and second layers of the cerebral cortex and the dentate gyrus was relatively strongly stained. Fiber-like structures were also stained in the CA3 region of hippocampus.     

The effects of serotonin on glucocorticoid receptor binding in rat raphe nuclei and hippocampal cells in culture

The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamus-pituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (+/-)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropiona mide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.     

Up-regulation of cerebral carbonic anhydrase by anoxic stress in piglets

The resuscitation of asphyxiated babies is associated with changes in cerebral protein synthesis that can influence the neurological outcome. Insufficient gas exchange results in rapid shifts in extracellular and intracellular pH. Carbonic anhydrase (CA) plays an important role in buffering acute changes in pH in the brain. We investigated whether asphyxia/re-ventilation influences the expression of cerebral CA isoforms (CA-II, CA-III and CA-IV) in anaesthetized newborn pigs. The cerebral cortex, hippocampus, cerebellum and retina were sampled, and prepared for either CA immunohistochemistry or CA immunoblotting from piglets subjected to asphyxia (10 min) followed by 2-4 h of re-ventilation, and also from normoxic controls. The CA immunoreactivity (IR) of all the isoforms studied was weak in the controls, apart from staining of a few oligodendrocytes in the subcortical white matter, some astrocytes in the superficial layer of the cerebral cortex, the cerebellar Purkinje cells and the retinal Müller cells that possessed moderate CA-II IR. However, asphyxia induced a marked increase in the CA IR of all isoforms in all the cerebral regions investigated and the retina after 4 h of survival. The pyramidal cells of the frontal cortex and hippocampus displayed the most conspicuous increase in CA IR. Immunoblotting confirmed increased levels of all the CA isoenzymes. We conclude that raised CA levels after asphyxia may contribute to the compensatory mechanisms that protect against the pathological changes in the neonatal CNS.     

High-affinity glycine and glutamate transport in pig forebrain white and gray matter: a quantitative study

High-affinity uptake of glycine and glutamate modulates glutamatergic neurotransmission in gray matter. N-Methyl-D-aspartate (NMDA) receptors were recently described on white matter oligodendrocytes, therefore uptake of glutamate and glycine in white matter may also modulate NMDA receptor function. We found that glycine uptake in white structures of pig forebrain (corpus callosum, fimbria, subcortical pyramidal tracts, and occipital subcortical white matter) was similar to that in gray structures (frontal and temporal cortices and hippocampus), and that it was sensitive to sarcosine, a GLYT1 inhibitor (IC(50) 15 microM). Glutamate uptake in white matter was approximately 10% of that in gray; it was sensitive to dihydrokainate, an EAAT2 inhibitor. The levels of glycine and its precursor serine were similar in white and gray matter: approximately 2 and 1 nmol/mg tissue, respectively. The white matter level of glutamate was approximately 7.6 nmol/mg tissue, or approximately 74% of gray matter levels. The activity of serine hydroxymethyl transferase, which converts serine into glycine, was similar in white and gray matter (11-18 pmol/(mg tissue)min), whereas the white matter activity of phosphate-activated glutaminase, which converts glutamine into glutamate, was approximately 100 pmol/(mg tissue)min, or approximately 34% of gray matter activity. The white matter activity of glutamine synthetase, the glial enzyme that converts glutamate into glutamine, was 20-40 nmol/(mg tissue)min in neocortex and 5-6 nmol/(mg tissue)min in white matter. The data show that forebrain white matter is equipped to regulate extracellular levels of glycine and glutamate, functions that may modulate white matter NMDA receptor function.     

Expression of carbonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic acid induced status epilepticus

Carbonic anhydrases (CAs) are important enzymes in the central nervous system (CNS), where they participate in regulating cerebrospinal fluid (CSF) secretion, blood-brain barrier and glial cell function. Using RT-PCR we found CA XII mRNA in rat and mouse brain. Cloning of rat CA XII revealed 94% homology with the mouse CA XII. To map the putative functional roles of different CAs, we studied the expression and localization of CA II, CA IV, CA VII, CA-related protein (CA-RP) VIII and CA XII mRNAs in rat brain after kainic acid induced epileptic seizures using Northern blot analysis and in situ hybridization. The expression of CA IV, CA VII and CA-RP VIII was somewhat similar: they were expressed in the cortex, hippocampus and midbrain structures and their expression did not change after the kainic acid treatment. The expression of CA II was concentrated in the white matter structures, which is in line with the preferential expression of CA II in the oligodendrocytes. High levels of CA II mRNA were also detected in the choroid plexus. Surprisingly, CA II was induced 3-12 h after seizures in the vulnerable CA1 region. CA XII was expressed in dentate granule cells, cortex and choroid plexus. Kainic acid stimulated CA XII expression throughout the cortical layer I. The observed hippocampal induction of CA II may indicate a pro-apoptotic and/or epileptogenic role of CA II after prolonged seizures. The physiological significance of the observed cortical induction of CA XII remains obscure. Cytosolic CA II is known to participate in CSF secretion, and the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme.