Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive

Glia show marked heterogeneity in terms of electrophysiology in the developing brain, and two major types can be identified based on GFAP or NG2 expression. However, it remains to be determined if such an electrophysiological diversity holds for the adult brain and how GFAP and NG2 lineage glia are associated with different electrophysiological phenotypes during the course of development. To address these fundamental questions, we performed in situ whole cell recording from morphologically identified glia from the rat hippocampal CA1 region from postnatal (P) days 1-106 and double-stained postrecorded cells with GLAST and NG2 antibodies. We found glia express mostly voltage-gated outward K(+) currents and also have inward Na(+) currents in the newborn (P1-P3), but these are no longer present after P22. They consist equally of GLAST(+) and NG2(+) cells in the newborn, but are mainly NG2(+) in juvenile animals (P4-P21). Glia showing voltage-gated outward and inward K(+) currents are also present at P1, peak at P5 and decline to a stationary level of approximately 10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive.     

Regional difference of reactive astrogliosis following traumatic brain injury revealed by hGFAP-GFP transgenic mice

Reactive astrocytes greatly influence the wound healing and neuronal regeneration processes following brain injury. However, the origin and fate of reactive astrocytes appear to be different depending on the type, severity and duration of brain injury. Using the cryogenic traumatic brain injury model, here we comprehensively addressed the regional differences of reactive astrocytes in the injured cortex. In the proximal region of injury site, NG2-expressing and cytoplasmic Olig2-labeled cells were densely localized 3 days after the injury. Next to this proximal layer, most of reactive astrocytes did not express NG2 but exhibited radial glia-like shape with elongated processes. Accordingly, they expressed the progenitor or radial glial markers, such as vimentin, brain lipid binding protein (BLBP) and the green fluorescent protein (GFP) under the control of the human GFAP (hGFAP) promoter. However, only few glial fibrillary acidic protein (GFAP) expressing astrocytes were found in this layer. Distal to the injury site, most of astrocytes strongly expressed GFAP with hypertonic morphology. At day 15 after injury, all layers expressing GFAP and other marker expressions disappeared, indicating the termination of reactive astrogliosis. Taken together, our data suggest that reactive astrogliosis occurs in a regionally segregated manner in the early phase of brain injury.     

Intrinsic Astrocyte Heterogeneity Influences Tumor Growth in Glioma Mouse Models

The influence of cellular origin on glioma pathogenesis remains elusive. We previously showed that mutations inactivating Rb and Pten and activating Kras transform astrocytes and induce tumorigenesis throughout the adult mouse brain. However, it remained unclear whether astrocyte subpopulations were susceptible to these mutations. We therefore used genetic lineage tracing and fate mapping in adult conditional, inducible genetically engineered mice to monitor transformation of glial fibrillary acidic protein (GFAP) and glutamate aspartate transporter (GLAST) astrocytes and immunofluorescence to monitor cellular composition of the tumor microenvironment over time. Because considerable regional heterogeneity exists among astrocytes, we also examined the influence of brain region on tumor growth. GFAP astrocyte transformation induced uniformly rapid, regionally independent tumor growth, but transformation of GLAST astrocytes induced slowly growing tumors with significant regional bias. Transformed GLAST astrocytes had reduced proliferative response in culture and in vivo and malignant progression was delayed in these tumors. Recruited glial cells, including proliferating astrocytes, oligodendrocyte progenitors and microglia, were the majority of GLAST, but not GFAP astrocyte‐derived tumors and their abundance dynamically changed over time. These results suggest that intrinsic astrocyte heterogeneity, and perhaps regional brain microenvironment, significantly contributes to glioma pathogenesis.     

Expression of amyloid precursor protein-like molecule in astroglial cells of the subventricular zone and rostral migratory stream of the adult rat forebrain

In adult mammals, new neurons in the subventricular zone (SVZ) of the lateral ventricle (LV) migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they mature into local interneurons. Using a monoclonal antibody for the beta-amyloid precursor protein (APP) (mAb 22C11), which is specific for the amino-terminal region of the secreted form of APP and recognizes all APP isoforms and APP-related proteins, immunoreactivity was detected in specific subpopulations of cells in the SVZ and RMS of the adult rat forebrain. In the SVZ, APP-like immunoreactivity was detected in the ependymal cells lining the LV and some of the subependymal cells. The latter were regarded as astrocytes, because they were positive for the glial markers, S-100 protein (S-100) and glial fibrillary acidic protein (GFAP). APP-like immunoreactive astrocytes exhibited strong labelling of the perinuclear cytoplasm and often possessed a long, fine process similar to that found with radial glia. The process extended to an APP-like immunoreactive meshwork in the RMS that consisted of cytoplasmic processes of astrocytes forming 'glial tubes'. Double-immunofluorescent labelling with a highly polysialylated neural cell adhesion molecule (PSA-NCAM) confirmed that the APP-like immunoreactive astrocytes in the SVZ and meshwork in the RMS made close contact with PSA-NCAM-immunopositive neuroblasts, suggesting an interaction between APP-containing cells and neuroblasts. This region of the adult brain is a useful in vivo model to investigate the role of APP in neurogenesis.     

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Summary Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.     

成人侧脑室下区Nestin阳性细胞的免疫组化观察

目的　探讨成人侧脑室下区 (SVZ)Nestin阳性细胞的分布及其化学特点。方法　采用 3 3 1B、10C2、Rat4 0 1、GFAP、NSE和viminten等神经细胞标志物对成人SVZ区进行免疫组织化学研究。结果　SVZ区存在较多Nestin反应阳性细胞 ,GFAP免疫阳性细胞及较少的NSE免疫阳性细胞。Nestin免疫反应阳性细胞可分为两类 ,一类为卵圆形 ,胞体较大 ,少有纤维突起 ;一类为星形 ,胞体较小 ,发出多支放射状的纤维突起。Nestin免疫阳性细胞均不与GFAP及NSE发生交叉反应。成人SVZ区细胞未见有viminten的表达。结论　成人SVZ区可能存在神经前体细胞和星形胶质样Nestin免疫阳性细胞。     

An immunohistochemical study of the topography and cellular localization of three neural proteins in the sheep nervous system.

The peroxidase-anti-peroxidase (PAP) method was used to determine the topography and cellular localization of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and carbonic anhydrase II (CAII) in the central nervous system (CNS), dorsal root ganglia and dorsal and ventral spinal nerve roots of the sheep. Parallel studies of mouse brain provided comparative data. Several fixatives were compared for their relative merits in preserving marker protein expression: GFAP was well preserved irrespective of the fixative employed; MBP was best preserved in formal sublimate and CAII was best preserved in Carnoy's fluid. In sheep, GFAP expression was seen in protoplasmic and fibrous astrocytes, Bergmann glial cells, a proportion of ependymal cells, amphicytes of spinal ganglia and in a proportion of presumed Schwann cells of dorsal and ventral spinal nerve roots. MBP expression was seen in mature and developing myelin sheaths of the central nervous system and in the cytoplasm of sparse myelinating oligodendroglia of the sub-cortical white matter of the cerebrum. CAII expression was seen in choroid plexus epithelium in all ages of sheep studied and, in a young lamb and an adult sheep, in glia and neuropil of ventral horn grey matter of the spinal cord and in the cytoplasm of white matter glia, presumed fibrous astrocytes, throughout the CNS. Compared with sheep brain, mouse brain showed the following differences in marker protein localization. GFAP was weakly expressed by protoplasmic astrocytes and not expressed in ependyma, oligodendroglia expressing intracytoplasmic MBP were frequent and widespread in neonatal mouse brain, CAII was expressed in myelin and oligodendroglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of adult neural stem cells and their relation to brain tumors

The adult mammalian brain contains neural stem cells that are capable of generating new neurons and glia over the course of a lifetime. Neural stem cells reside in 2 germinal niches, the subventricular zone (SVZ) and the dentate gyrus subgranular zone. These primary progenitors have been identified in their niche in vivo; these cells have characteristics of astrocytes. Recent studies have shown that adult SVZ stem cells are derived from radial glia, the stem cells in the developing brain, which in turn are derived from the neuroepithelum, the earliest brain progenitors. Thus, SVZ stem cells are a continuum from neuroepithelium to radial glia to astrocytes, and are contained within what has been considered the lineage for astrocytes. However, it seems that only a small subset of the astrocytes present in the adult brain have stem cell properties. Recent findings have shown that SVZ stem cell astrocytes express a receptor for platelet-derived growth factor (PDGF), suggesting that the ability to respond to specific growth factor stimuli, such as PDGF, epidermal growth factor and others, may be unique to these stem cell astrocytes. Intriguingly, activation of these same signaling pathways is widely implicated in brain tumor formation. Since the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for transformation, the adult neural stem and/or progenitor cells may be likely candidates for the brain tumor cell of origin. Indeed, activation of the PDGF or epidermal growth factor pathways in adult neural stem or progenitor cells confers tumor-like properties on these cells, lending support to this hypothesis.     

成年大鼠侧脑室下区星形胶质细胞的分离培养及纯化研究

成年哺乳动物侧脑室下区星形胶质细胞(AST)的神经干细胞特性研究是当前神经科学领域的热点,为了进一步了解AST生物学特性,体外培养是探讨AST细胞学特性的有力手段,而怎样通过体外培养得到高纯度的成年AST是亟待解决的问题。本研究以2.5月的成年大鼠侧脑室下区为实验材料进行AST培养,并对传统的培养方法进行改良。首先在AST培养基中增加B27添加剂,结合成年AST体外生长特性,采用低浓度血清(2.5%～5%)和血清浓度调整法对培养的AST进行纯化。然后采用GFAP免疫细胞化学染色方法对培养至14d的细胞进行鉴定及纯度检测,采用流式细胞仪检测B27添加剂对AST细胞周期的影响。结果显示:B27添加剂能有效促进体外培养的成年AST的生长和增殖,采用此方法培养的成年AST纯度达90%以上;B27添加剂与低浓度血清的组合以及根据细胞特性及时调整血清浓度是一种有效的纯化成年AST方法。本方法的建立可望为AST的神经干细胞特性研究提供理想的细胞模型。     

Increased neurogenesis and astrogenesis from neural progenitor cells grafted in the hippocampus of GFAP-/- Vim-/- mice

After neurotrauma, ischemia, or neurodegenerative disease, astrocytes upregulate their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin (Vim), and nestin. This response, reactive gliosis, is attenuated in GFAP(-/-)Vim(-/-) mice, resulting in the promotion of synaptic regeneration after neurotrauma and improved integration of retinal grafts. Here we assessed whether GFAP(-/-)Vim(-/-) astrocytes affect the differentiation of neural progenitor cells. In coculture with GFAP(-/-)Vim(-/-) astrocytes, neural progenitor cells increased neurogenesis by 65% and astrogenesis by 124%. At 35 days after transplantation of neural progenitor cells into the hippocampus, adult GFAP(-/-)Vim(-/-) mice had more transplant-derived neurons and astrocytes than wild-type controls, as well as increased branching of neurite-like processes on transplanted cells. Wnt3 immunoreactivity was readily detected in hippocampal astrocytes in wild-type but not in GFAP(-/-)Vim(-/-) mice. These findings suggest that GFAP(-/-)Vim(-/-) astrocytes allow more neural progenitor cell-derived neurons and astrocytes to survive weeks after transplantation. Thus, reactive gliosis may adversely affect the integration of transplanted neural progenitor cells in the brain. Disclosure of potential conflicts of interest is found at the end of this article.     

成年大鼠去皮层血管引起前脑室下区祖细胞增殖迁移并形成迁移路至损伤部位(一)祖细胞增殖迁移的部位在背外侧脑室下区

成年哺乳动物脑室下区(SVZ)富有神经干细胞、神经细胞祖细胞和胶质细胞祖细胞,它们能生成新的神经细胞、星状胶质细胞和少突胶质细胞。SVZ中的神经细胞祖细胞能形成切线形式的嘴侧迁移流(RMS)到嗅球,在嗅球分化成成熟的中间神经元。近年来证明成年动物实验性脑损伤和变性疾病都能引起SVZ细胞增生并能向非嗅球区迁移。本研究将成年大鼠一侧大脑皮层血管去除,15d和30d后取前脑作冠状及矢状连续切片,用BrdU和PCNA抗体显示前脑室下区正在分裂的细胞;用Tuj1抗体显示神经元祖细胞;用GFAP和vimentin抗体显示胶质细胞祖细胞。结果证明去除一侧皮层血管引起术侧及其对侧的背外侧脑室下区(dl-SVZ)的上述免疫反应阳性细胞明显增多,并向胼胝体迁移,在胼胝体内形成放射形式迁移路至损伤部位。本研究表明背外侧脑室下区的范围应包括背外侧角、外侧伸展和侧脑室上壁的SVZ,它们是切线形式和放射形式两种不同方向的迁移路祖细胞的共同源地。     

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of adult Podarcis sicula (Squamata, Lacertidae)

The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula . GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star-shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.     

Expression of glial fibrillary acidic protein, actin, fibronectin and factor VIII antigen in human astrocytomas

Frozen sections and cell cultures of 50 human astrocytomas, fetal and adult human brain were examined for immunofluorescence reactivity with antisera to glial fibrillary acidic protein (GFAP), actin, fibronectin and factor VIII antigen. In frozen tissue sections GFAP expression was restricted to normal and neoplastic astrocytes while fibronectin and factor VIII antigen were localized to blood vessels. In primary cell culture, 80-100% neoplastic astrocytes expressed GFAP but not fibronectin or factor VIII antigen while actin was present as diffuse cytoplasmic staining of the cell body and cell processes. By the 5th-6th passage in vitro, GFAP immunoreactivity was lost while fibronectin and actin cables were prominently expressed. Factor VIII antigen remained negative throughout serial subculture. In double fluorochrome experiments, GFAP positive cells did not express fibronectin or actin cables, while GFAP negative cells expressed fibronectin and had prominent actin cables. Our results suggest a change in population of astrocytoma cells with increasing passage in vitro, reflecting either an overgrowth of tumour glioblasts or dedifferentiation of tumour astrocytes.     

Changes in ganglioside composition and morphological features during the development of cultured astrocytes from rat brain

Changes in ganglioside content over a period of days were examined in astrocytes obtained via cell passage from rat cerebral cortex. Thin-layer chromatography revealed that, in the astrocytes, ganglioside GM1 was absent, the predominant ganglioside being GM3. Also, an increased GD3 content in long-term astrocyte cultures was detected. The morphological features of astrocytes were also studied using immunoperoxidase staining. Astroglial features were characterized by high levels of glial fibrillary acidic protein (GFAP) and vimentin, which are the major intermediate-filament proteins present in astrocytes at an early culture stage. In long-term-cultured (greater than 7 months) astrocytes, vimentin and GFAP were increased in process-bearing cells. Ganglioside GD3 recognized by R24 monoclonal antibody was also expressed in these cells. These results suggest that the increase of ganglioside GD3 in long-term-cultured astrocytes may be related to the appearance of multistellate cells showing strong reactivity against GFAP and vimentin during development over a specified period in culture.     

Immunocytochemical demonstration of glial fibrillary acidic protein in mouse tanycytes

Summary Immunohistochemical techniques were used to stain for the astrocytespecific glial fibrillary acidic protein (GFAP) in the cells lining the third ventricle of the developing and mature mouse brain. Before birth immunoreactive tanycytes were only observed in the infundibular recess of the median eminence, where they could first be seen at embryonic day 17. They possessed long processes running towards the ventral surface of the brain. During the early postnatal period GFAP-positive tanycytes gradually appeared throughout the third ventricle, although the ependymal cells themselves remained unstained. The tanycytes retained their immunoreactivity for anti-GFAP serum in the adult, and were also evident in the adult rat third ventricle. It is suggested that the presence of GFAP in these specialised cells of the third ventricle indicates that they, the transient radial glia of the developing cerebral cortex, the persistent Bergmann glia of the cerebellum, similar astrocytes with radial processes in the hippocampal dentate gyrus and conventional astroglia are all closely related cell types.     

Immature-like astrocytes are associated with dentate granule cell migration in human temporal lobe epilepsy

In human temporal lobe epilepsy, a dispersion of dentate granule cells is frequently described in adults who had an early risk factor. To elucidate the role of glia in this phenomenon, we investigated neuronal dispersion, astrocyte organization and expression of intermediate filaments of mature and immature astrocytes (i.e. glial fibrillary acidic protein (GFAP) and vimentin, respectively) in seven subjects with early febrile seizures (F(+)) and five subjects with other etiologies than febrile seizures (F(-)). Compared to F(-) patients, a majority of F(+) subjects showed neuronal dispersion and vimentin expression in radial glia. However, in two patients with the maximal dispersion, radial processes expressed only GFAP. We suggest that granule cell migration that occurs in adult epileptic focus results from the transient occurrence of immature-like glia throughout the granular layer.     

Differential expression of Hoxa-2 protein along the dorsal-ventral axis of the developing and adult mouse spinal cord.

We have used synthetic oligopeptides derived from the coding sequence of the murine Hoxa-2 protein to produce polyclonal antibodies that specifically recognize the Hoxa-2 recombinant protein. Immunohistochemical studies reveal a distinct pattern of spatial and temporal expression of Hoxa-2 protein within the mouse spinal cord which is concomitant with the cytoarchitectural changes occurring in the developing cord. Hoxa-2 protein is predominantly detected in the nuclei of cells in the ventral mantle region of 10-day-old mouse embryos. Islet-1, a marker for motor neurons was also shown to be co-localized with Hoxa-2 in nuclei of cells in this region. As development progresses from 10-days to 14-days of gestation, Hoxa-2 protein expression gradually extends to the dorsal regions of the mantle layer. The Hoxa-2 protein expression pattern changes at 16-days of embryonic development with strong expression visible throughout the dorsal mantle layer. In 18-day-old and adult mouse spinal cords, Hoxa-2 protein was expressed predominantly by cells of the dorsal horn and only by a few cells of the ventral horn. Double labeling studies with an antibody against glial fibrillary acidic protein (GFAP, an astrocyte-specific intermediate filament protein) showed that within the adult spinal cord, astrocytes rarely expressed the Hoxa-2 protein. However, Hoxa-2 and GFAP double-labeled astrocytes were found in the neopallial cultures, although not all astrocytes expressed Hoxa-2. Hoxa-2 expressing oligodendrocyte progenitor cells were also identified after double-labeling with O4 and Hoxa-2 antibodies; although cells in this lineage that have begun to develop a more extensive array of cytoplasmic processes were less likely to be Hoxa-2 positive. The early pattern of Hoxa-2 protein expression across transverse sections of the neural tube is temporally and spatially modified as each major class of neuron is generated. This congruence in the expression of the Hoxa-2 protein and the generation of neurons in the cord suggests that the Hoxa-2 protein may contribute to dorsal-ventral patterning and/or to the specification of neuronal phenotype. Dev Dyn 1999;216:201-217.     

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.