NUMBER OF PURKINJE CELLS AND BERGMANN ASTROCYTES IN RATS WITH CCl4-INDUCED LIVER DISEASE

The nuclei of Purkinje cells and Bergmann astrocytes were counted on sagittal sections from cerebellum and the length of stratum gangliosum was measured in rats with CCl₄-induced liver disease, using an electronic image analyzer. After 8 weeks of CCl₄-administration a reduction was found in the number of Purkinje cells, many of which showed homogenizating changes. Ten weeks after termination of the administration period the number of Purkinje cells was reduced by 12 per cent. The number of Bergmann astrocytes remained significantly increased after 8 weeks of CCl₄-administration (max. 20 per cent). The changes of Purkinje cell and Bergmann astrocyte density developed during the period of severe liver necrosis, whereas only minor changes were found in the ensuing period of liver "cirrhosis". In the perfusion fixed specimens, the Bergmann astrocyte nuclei increased in volume up to 65 per cent and immersion fixed brains showed typical Alzheimer type II nuclear changes. The impact of the increased plasma ammonia concentration on the astrocytes is discussed.     

Glial and nerve cell changes in rats with porto-caval anastomosis

Summary Nuclear size and density were determined in brain regions with different glial—neurone composition in rats up to 35 weeks after porto-caval anastomosis.In the white matter, i.e. corpus callosum, both the total cell count and the percentage of astrocytes and oligodendrocytes were unchanged.In the corpus striatum, where the glial/neurone ratio is about 1, the number of nuclei registered as astrocytes increased, and after 35 weeks astrocytes comprised 29% of glial cells (compared with 15% in controls). However, the number of oligodendrogial nuclei decreased simultaneously, leaving the total glial number unchanged. In the animals with longest experimental period there was a 15% loss of neurones.In a region with higher glial/neurone ratio, i.e. the Purkinje cell layer, the neurones showed a similar reduction, whereas the number of Bergmann astrocyte nuclei increased less than striatal astrocytes.A small group of animals with pronounced signs of encephalopathy had a higher loss of neurones and, furthermore, the glial number in corpus striatum and callosum was reduced, due to loss of oligodendrocytes.Despite the use of perfusion fixation, the size of astrocyte nuclei increased, this was reversible, as only slight changes were seen after 35 weeks.A possible explanation of the increase in astrocyte nuclear count and decrease in oligodendroglial count could be that nuclei normally considered to the oligodendroglial are transformed into nuclei with morphological characteristics of astrocytes.     

GLIAL CELL REACTIONS IN RATS WITH HYPERAMMONIEMIA INDUCED BY UREASE OR PORTO-CAVAL ANASTOMOSIS

The density and size of astrocyte, oligodendrocyte and neurone nuclei were determined in the corpus striatum of rats with urease-induced hyperammoniemia (plasma NH₄+-concentration about 800 μmol/l). No changes in the number of neurone nuclei were found. After 4 days the density of astrocyte nuclei increased from 13 to 23 per cent of the glial nuclei. However, the number of oligodendrocyte nuclei decreased correspondingly and the total glial nuclear number remained constant. The number of astrocyte nuclei was normalized 1 week after the 4-day period of hyperammoniemia. Moreover, intracerebral injections of colchicine at different times of the experimental period revealed no mitoses, indicating that no astrocyte divisions took place in pure hyperammoniemia. The astrocyte nuclei were of normal size in the urease animals in contrast to the animals with porto-caval anastomosis (PCA) which showed enlarged astrocyte nuclei. Thus, hyperammoniemia caused a reversible transformation of glial nuclei, but no real proliferation. A comparison of the glial reactions 4 days after a brain lesion showed the same frequency of astrocyte mitoses in control and urease animals but a higher incidence of mitoses in the PCA animals. The number of Alzheimer type I astrocytes was the same in control and PCA animals, whereas no such cells were found in the urease animals, indicating that this form of hyperammoniemia did not lead to arrest of astrocyte metaphases with subsequent formation of Alzheimer type I cells.     

MORPHOMETRIC STUDIES OF RAT GLIAL CELL ULTRASTRUCTURE AFTER UREASE-INDUCED HYPERAMMONAEMIA

The ultrastructure of astrocytes and oligodendrocytes was investigated in hyperammonaemic rats injected daily with urease for 4 days. Glial cells were randomly photographed and magnified x28 000. Cell and nuclear sizes were estimated by planimetry and mitochondrial size and density were measured by image analysis. After 4 days of hyperammonaemia the astrocyte cytoplasmic area was increased by 46%. Mitochondrial area was increased by 20%, but after correction for cytoplasmic oedema the number and size of mitochondria were not significantly increased. The nuclear and cytoplasmic areas of oligodendrocytes were unchanged. The mitochondria of oligodendrocytes were small in the hyperammonaemic group and so was their percentage area to cytoplasmic area, but their numbers were unchanged. It was concluded that hyperammonaemia induces astrocyte oedema and increases the astrocyte mitochondrial content. These findings support the assumption that the astrocytes are the active cells in the brain metabolism of ammonia. The decrease in oligodendrocyte mitochondrial content could be considered a point against an active function of oligodendrocyte mitochondria in ammonia metabolism in hyperammonaemia.     

Endothelins Promote the Activation of Astrocytes in Rat Neostriatum through ETB Receptors

The effects of endothelin (ET)-3 and an ET_B receptor agonist on astrocytic activation in rat caudate putamen were examined by an immunohistochemical staining of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes. A single injection of 40 pmol ET-3 into rat caudate putamen increased the number of GFAP positive cells compared to that in the contralateral saline-injected side. Ala^1,3,11,15-ET-1 (40 pmol), an ET_B receptor agonist, also increased the number of striatal GFAP positive cells. The increases in GFAP positive cells were maximum (about 150% of the control side) in 1–2 weeks after injections of the ET_s, and then reduced in 4 weeks. A continuous infusion of BQ788, an ET_B receptor antagonist (23 nmol/day), into the lateral ventricle of the cerebrum antagonized the effect of Ala^1,3,11,15-ET-1, while 80788 also reduced the number of GFAP positive cells in saline-injected caudate putamen. Intrastriatal injection of 40 pmol Ala^1,3,11,15-ET-1 did not affect the number of cells stained by B₄ isolectin from Griffonia simplicifolia , which labels activated microglia/macrophages. Intraperitoneal administration of 5 mg/kg per day chloroquine and 0.2 mg/kg per day colchicine did not affect the action of Ala^1,3,11,15-ET-1. These results suggest that activation of ET_B receptors is involved in the induction of reactive astrocytes.     

Morphometry of astrocyte and oligodendrocyte ultrastructure after portocaval anastomosis in the rat

Summary The ultrastructure of astrocytes and oligodendrocytes was investigated in rats 10 days, 30 days, and 10 weeks after portocaval anastomosis (PCA). Cell and nuclear sizes were measured by planimetry on randomly sampled cells magnified×24,000. The volume fractions of mitochondria, glia fibrils, and lipofuscin granules were measured in astrocytes by electronic image analysis. The mitochondrial profile area distribution and oligodendrocyte mitochondrial content were likewise estimated. All PCA animals had an increased astrocyte cell and cytoplasmic area, and after correction for cytoplasmic edema all groups had an enhanced mitochondrial fraction and mitochondrial number. The mitochondrial sizes were increased in all PCA groups. The mitochondrial profile area distribution curves did not suggest more than one group of mitochondria. All PCA groups had increased fractions of lipofuscin granules and glia fibrils. The oligodendrocytes had a slight fall in cell, nuclear, and cytoplasmic area after 30 days of shunting, and the mitochondrial fraction was diminished. After 10 weeks of PCA, all changes were reversed to normal values. It is concluded that the astrocytes are the active cells in the brain metabolism of ammonium. The oligodendrocytes seem to be dependent on neuronal integrity and do not contribute to the brain ammonium metabolism. The increase in astrocyte lipofuscin granules content may be explained by a beginning neuronal loss.Abbreviation (PCA) Portocaval anastomosis     

Astrocytes in the hypoglossal nuclei of sudden infant death syndrome (SIDS) infants: a quantitative study

It has been suggested that brain stem hypoxia or ischaemia underlies the sudden infant death syndrome (SIDS), but previous reports of astrocytosis in the brain stems of SIDS infants have been contradictory. A volumetric quantitative technique was, therefore, developed to compare astrocyte numbers and sizes in the hypoglossal nuclei of SIDS and control infancts. In 12 SIDS and eight control infants, serial sagittal sections were taken through the hypoglossal nucleus and every tenth section through the h ypoglossal nucleus and every tenth section was stained for glial fibrillary acidic protein. Astrocytes were counted in the central 4% of a grid stepped throughout the hypoglossal nucleus, and the heights of 100 astrocyte nuclei were measured with a mocrocator. Astrocyte number, corrected for section thickness and nuclear height, was divided by the volume of the hypoglossal nucleus to calculate astrocyte density. Numbers of astrocytes did not differ significantly between SIDS (mean number 44 729, SD 12 096) and control (mean number 46 562, SD 11 060) infants. Astorcyte nuclear height did not differ significantly between groups (SIDS: mean height 3.98μm, SD 0.31). Astrocyte density was similar in SIDS (mean density 24 378 astrocyte/mm³, SD 6155) and control (mean density 23 978 astrocytes/mm³, SD 4031) infants. No quantitative evidence of astrocytosis was found in the hypoglossal nuclei of SIDS infants. This implies that SIDS infants die without previous episodes of hypoxia/ischaemia severe enough to damage the brain stem.     

Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency

Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH₄⁺-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH₄⁺ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH₄Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4⁺ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH₄⁺ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH₄⁺ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood–brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.     

Insulin-like growth factor I promotes cell proliferation and oligodendroglial commitment in rat glial progenitor cells developing in vitro

We investigated the mechanisms by which insulin-like growth factor I (IGF-I) acts to increase the number of oligodendrocytes that develop in cultures of cells explanted from perinatal rat cerebrum. Fluorescence-activated cell sorting was used to isolate bipotential A2B5-positive oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, which were then inoculated as single cells into microculture wells containing feeder layers of X-irradiated type 1 astrocytes. Addition of 100 ng/ml IGF-I to the culture medium increased the growth rate and the ultimate size reached by the resulting clones during the 18-day experimental period. Moreover, 75-80% of the cells in the IGF-I-treated clones differentiated into galactocerebroside (GC)-positive oligodendrocytes, whereas only 25-30% became oligodendrocytes in the absence of IGF-I. IGF-I did not increase the number of type 2 astrocytes that developed in the clones. IGF-I appeared to have the greatest effect on growth and differentiation at a stage when the majority of the cells in the clones were at an intermediate stage of development, characterized by the expression of A2B5 and O4 glycolipid antigens but not GC. Analysis of the effects of IGF-I on O4-positive, GC-negative intermediate precursor cells revealed a two to fivefold increase in the number of cells that incorporated 3H-thymidine into their DNA during a 5-h pulse. Moreover, IGF-I increased the number of cell sorter-purified O4-positive cells that developed into oligodendrocytes 4-8 days later. Therefore, IGF-I acts in two different ways to promote oligodendrocyte development: It promotes proliferation of precursor cells in the O-2A lineage, and it induces precursors to become committed to develop into oligodendrocytes.     

Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole‐cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction‐permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin‐Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47‐deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47‐double‐deficient mice. Cx47‐ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43‐deficient animals, oligodendrocyte‐astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30‐double deficient mice, oligodendrocyte‐to‐astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network. © 2010 Wiley‐Liss, Inc.     

Ca2+ Channel Expression in the Oligodendrocyte Lineage

The development of oligodendrocytes from their precursor cells through different developmental stages can be studied in vitro. These stages can be distinguished by specific monoclonal antibodies and by a characteristic K⁺ channel profile. In this study we demonstrate that the occurrence of Ca²⁺ currents also undergoes marked changes during the development of mouse oligodendrocytes. Immature precursor cells which can develop into astrocytes or oligodendrocytes expressed two different types of voltage-activated Ca²⁺ channels. The expression of Ca²⁺ channels in precursor cells was strongly correlated with the expression of Na⁺ channels. When cells started to express the O1 antigen and were committed to the oligodendrocyte lineage, Ca²⁺ and Na⁺ currents could no longer be detected. Large Ca²⁺ currents were, however, recorded later in the development of the oligodendrocytes, correlated with the expression of the O10 antigen. The Ca²⁺ channels were classified as high and low voltage-activated Ca²⁺ channels according to their range of activation, and are further described by their kinetic and pharmacological properties.     

Glycine- and GABA-activated Currents in Identified Glial Cells of the Developing Rat Spinal Cord Slice

In the neonatal rat spinal cord, four types of glial cells, namely astrocytes, oligodendrocytes and two types of precursor cells, can be distinguished based on their membrane current patterns and distinct morphological features. In the present study, we demonstrate that these cells respond to the inhibitory neurotransmitters glycine and GABA, as revealed with the whole-cell recording configuration of the patch-clamp technique. All astrocytes and glial precursor cells and a subpopulation of oligodendrocytes responded to glycine. The involvement of glycine receptors was inferred from the observation that the response was blocked by strychnine and that the induced current reversed close to the Cl^- equilibrium potential. GABA induced large membrane currents in astrocytes and precursor cells while oligodendrocytes showed only small responses. The GABA-activated current was due to the activation of GABA_A receptors since muscimol mimicked and bicuculline blocked the response; moreover, the reversal potential was close to the Cl^- equilibrium potential. Besides the increase in a Cl^- conductance, GABA^A receptor activation also induced a block of the resting K⁺ conductance, as observed previously in Bergmann glial cells. Our experiments show that while glial GABA_A receptors are found in many brain regions and the spinal cord, glial glycine receptors have so far been detected only in the spinal cord. The restricted coexpression of glial and neuronal glycine receptors in a defined central nervous system grey matter area implies that such glial receptors may be involved in synaptic transmission.     

Fractionation and enrichment of oligodendrocytes from developing human brain.

Enriched cultures of human oligodendrocytes were obtained from fetal brain specimens between 16 and 21 gestational weeks. Brain cells were separated over a Percoll density gradient and collected as two fractions with initial relative densities of approximately 1.035 g/ml and 1.102 g/ml, for fractions 1 and 2, respectively. After separation, 58.3 and 67.7% of the cells in fractions 1 and 2, respectively, were labeled by the antibody O4 that recognizes immature oligodendrocytes. A total of 15.5 and 29.4% of the cells in fractions 1 and 2, respectively, were positive for tubulin-beta(III), a marker for neurons but none of the freshly isolated cells were positive for glial fibrillary acidic protein (GFAP), a protein associated with astrocytes in the central nervous system. When the fractionated cells were cultured on poly-ornithine coated coverslips for 3 days and processed for immunocytochemistry, the percentage of O4+ oligodendrocytes decreased to less than 4% whereas GFAP+ cells increased to 1.8 and 12.4% for fractions 1 and 2 respectively. The percentage of tubulin-betaIII+ cells increased to 46 and 61% in cultures from the two Percoll fractions. This increase is probably due to the decrease in the number of oligodendrocytes. To avoid the loss of oligodendrocytes, cells were cultured as free-floating aggregates in the presence of 20 ng/ml of fibroblast growth factor-2 for 2 weeks. The resultant cultures became enriched for oligodendrocytes as demonstrated by cellular morphology and by positive O4 labeling. The method described here provides a means of obtaining enriched cultures of immature human oligodendrocytes for developmental and transplantation studies.     

Brain-Derived Neurotrophic Factor in Astrocytes, Oligodendrocytes, and Microglia/Macrophages after Spinal Cord Injury

Recent studies suggest that the injured adult spinal cord responds to brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) with enhanced neuron survival and axon regeneration. Potential neurotrophin sources and cellular localization in spinal cord are largely undefined. We examined glial BDNF localization in normal cord and its temporospatial distribution after injury in vivo. We used dual immunolabeling for BDNF and glial fibrillary acidic protein (GFAP) in astrocytes, adenomatous polyposis coli tumor suppressor protein (APC) for oligodendrocytes or type III CDH receptor (OX42) for microglia/macrophages. In normal cord, small subsets of astrocytes and microglia/macrophages and most oligodendrocytes exhibited BDNF-immunoreactivity. Following injury, the number of BDNF-immunopositive astrocytes and microglia/macrophages increased dramatically at the injury site over time. Most oligodendrocytes contained BDNF 1 day and 1 week following injury, but APC-positive cells were largely absent at the injury site 6 weeks postinjury. Glial BDNF-immunolabeling was also examined 10 and 20 mm from the wound. Ten millimeters from the lesion, astrocyte and microglia/macrophage BDNF-immunolabeling resembled that at the injury at all times examined. Twenty millimeters from injury, BDNF localization in all three glial subtypes resembled controls, regardless of time postlesion. Our findings suggest that in normal adult cord, astrocytes, oligodendrocytes, and microglia/macrophages play roles in local trophin availability and in trophin-mediated injury and healing responses directly within and surrounding the wound site.     

Transformation of postischemic perineuronal glial cells. I. Electron microscopic studies

The effects of cerebral ischemia on perineuronal glia were studied in the rat model of transient four-vessel occlusion. Striatum containing irreversibly injured neurons and paramedian cerebral cortex containing reversibly injured neurons were prepared for electron microscopy at intervals of 3 min up to 24 h following ischemia. Perineuronal astrocytes showed cytoplasmic swelling and configurational changes in and pleomorphism of mitochondria similar to those described previously in parenchymal astrocytes in this model. Dark oligodendroglia showed only transient swelling of cisterns of Golgi apparatus and endoplasmic reticulum. However, medium-light oligodendrocytes significantly increased in size and accumulated microtubules and tubovesicular profiles in the cytoplasm. Reactive glia with features of both oligodendrocytes and astrocytes appeared at 15 min. A sharp drop in the number of perineuronal medium-light oligodendrocytes occurred at 3 h after ischemia and was accompanied by increased numbers of astrocytes and intermediate glia. Cortical glia showed similar changes that were milder and reversible. These studies suggest that certain perineuronal glia are transformed into reactive astrocytes in areas of ischemic neuronal necrosis, although current data are insufficient to determine if the transforming cells are astrocytes, light oligodendrocytes, or intermediate glia. Possible stimuli for these glial reactions include loss of or changes in neuronal trophic factors upon CNS glia or alterations in the interstitial fluid composition.     

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low-density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high-density culture without TH. This suggests that, in low-density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte-inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell-cell communications.     

Development of Neurons and Glial Cells in Cerebral Cortex,Cultured in the Presence or Absence of Bioelectric Activity: Morphological Observations

Chronic blockade of bioelectric activity (BEA) has been shown to increase neuronal cell death in tissue culture, but the effects of this treatment on non-neuronal cells have not been investigated. To determine which cell types are affected by chronic suppression of BEA, we investigated their morphological development in primary cultures of rat cerebral cortex, grown with or without the sodium channel blocker tetrodotoxin (TTX). Morphological development was monitored by phase-contrast microscopy and by immunofluorescent staining of markers specific for neurons (NSE, MAP2, B-50, and the 200 kD neurofilament protein), astrocytes (GFAP), oligodendrocytes (galactocerebroside), macrophages (ED-1) and fibroblasts (fibronectin). Neurons in control cultures steadily increased in size and elaborated a dense network of axons and dendrites during the first 3 weeks. Astrocytes proliferated strongly and formed a 'bottom-layer' on which other cells grew. Part of the astrocytes migrated into the peripheral area of the culture, but retracted to the centre after 14 days in vitro (DIV). Oligodendrocytes and macrophages also increased in number, but oligodendrocytes were completely lost by 28 DIV. After 3 weeks, axons that had grown into the periphery of the culture gradually retracted and/or degenerated, following the retracting astrocytes. Some of the neurons died after 21 DIV, but a large part persisted until 42 DIV. Upon TTX treatment from 5/6 DIV, cultures with few macrophages showed an increase in the proportion of necrotic nuclei at 14 and 21 DIV. The retraction of peripherally located fibres was accelerated by 3 - 4 days and their degeneration was augmented. Neuronal density decreased to zero between 21 and 42 DIV. Astrocytes showed a clear decrease in density from 28 DIV. Conversely, the density of macrophages was increased about two-fold from 14 DIV. These results indicate that both neurons and glia are affected by chronic TTX treatment.     

Glial reduction in amygdala in major depressive disorder is due to oligodendrocytes.

BACKGROUND: A previous study reported reductions in glial density and glia/neuron ratio in the amygdala of individuals with major depressive disorder (MDD), without a change in neuronal density. It is not known, however, whether this glial loss is due to astrocytes, oligodendrocytes, or microglia. METHODS: Tissue samples, equally from the right and left hemispheres, were obtained from subjects diagnosed with MDD (n = 8), bipolar disorder (BD) (n = 9), or no psychiatric disorders (n = 10). Sections were stained immunohistochemically for S-100beta (for astrocytes) and human leukocyte antigen (for microglia), and with the Nissl method. In Nissl-stained sections, oligodendrocytes have more compact, darker-stained nuclei, whereas astrocytes and microglia have larger, lighter-stained nuclei, with more granular chromatin. Neurons are larger, with a nucleolus and stained cytoplasm. The density of glia was determined with stereologic methods. RESULTS: The density of total glia and oligodendrocytes in the amygdala was significantly lower in MDD than in control subjects, but not significantly lower in BD compared with control subjects. The decreases were largely accounted for by differences in the left hemisphere. There was no significant decrease in astrocyte or microglia density in MDD or BD subjects. CONCLUSIONS: The glial cell reduction previously found in the amygdala in MDD is primarily due to oligodendrocytes.     

Bipotential glial progenitors are targets of neuronal cell line-derived growth factors

The growth-promoting activity of conditioned medium (CM) from the B104 CNS neuronal cell line was studied in glial cultures from neonatal rat brain. This CM at 33% (v/v; 8-12 micrograms protein/ml) produced large numbers of oligodendrocytes and multipolar glial progenitors after an 8 to 12-day treatment. At all times studied, cells of the oligodendrocyte/type 2 astrocyte (O-2A) lineage were increased due to CM-treatment, while type 1 astrocytes, microglia, and other cell types were not. Furthermore, we observed a large decrease in the percentage of oligodendrocytes in the O-2A lineage, suggesting a delay in differentiation of the progenitors. By 8 days in vitro (DIV), dose-dependent increases in numbers of galactocerebroside (GalC)-positive cells (oligodendrocytes) and A2B5-positive cells (immature oligodendrocytes and glial progenitors) occurred. In contrast, at 4 DIV only A2B5-positive cells were increased in a dose-dependent manner. The latter cells can differentiate primarily into oligodendrocytes or type 2 astrocytes depending on the culture conditions. Complement lysis studies confirmed that the A2B5-positive, but not the GalC-positive, population at 4 DIV was required for increases in oligodendrocytes to occur by 8 DIV. The [3H]thymidine labeling index of the A2B5-positive population also increased in response to CM in a dose-dependent manner, but the GalC-positive labeling index showed only small increases at 4 DIV and none at later times. Our results suggest that the delayed differentiation coupled with the selective stimulation of the bipotential glial progenitors produces the large increases in numbers of oligodendrocytes observed at 8-12 DIV.