Morphological and physiological interactions of NG2-glia with astrocytes and neurons

Models of central nervous system (CNS) function have historically been based on neurons and their synaptic contacts - the neuronal doctrine. This doctrine envisages glia as passive supportive cells. However, electrophysiological and imaging studies in brain slices show us that astrocytes, the most numerous cells in the brain, express a wide range of neurotransmitter receptors that are activated in response to synaptic activity. Furthermore, astrocytes communicate via calcium signals that are propagated over long distances by the release of 'gliotransmitters', the most abundant being adenosine triphosphate (ATP). This has led to the concept of the neuron-astroglial functional unit as the substrate of integration in the CNS. Recently, a novel glial cell type has been characterized by expression of the proteoglycan NG2. These NG2-glia receive presynaptic input from neurons and responds to neurotransmitters released at synapses. Now, studies on transgenic mice in which fluorescent proteins are specifically expressed by subclasses of glia are helping to address the question of where NG2-glia fit in the neuron-astroglial model of integrated brain function. NG2-glia, as well as astrocytes, have been shown to respond to neuronal and astroglial signals by raised intracellular calcium, which is a potential communications mechanism by which NG2-glia may be active partners in neuron-glial circuits. Moreover, a current concept of NG2-glia considers them to be 'neural stem cells' and an exciting prospect is that neuron-glial signalling may regulate the differentiation capacity of NG2-glia and their response to injury.     

Fate of neuron-glia synapses during proliferation and differentiation of NG2 cells

Progenitor cells expressing proteoglycan NG2 (also known as oligodendrocyte precursor cells or polydendrocytes) are widespread in the grey and white matter of the CNS; they comprise 8-9% of the total cell population in adult white matter, and 2-3% of total cells in adult grey matter. NG2 cells have a complex stellate morphology, with highly branched processes that may extend more than 100 μm around the cell body. NG2 cells express a complex set of voltage-gated channels, AMPA/kainate and/or γ-aminobutyric acid (GABA)(A) receptors, and receive glutamatergic and/or GABAergic synaptic input from neurons. In every region of the brain NG2 cells are found as proliferative cells, and the fraction of actively cycling NG2 cells is quite high in young as well as in adult animals. During development NG2 cells either differentiate into myelinating oligodendrocytes (and possibly also few astrocytes and neurons) or persist in the brain parenchyma as NG2 cells. This review highlights new findings related to the morphological and electrophysiological changes of NG2 cells, and the fate of synaptic input between neurons and NG2 cells during proliferation and differentiation of these cells in the neonatal and adult nervous system of rodents.     

Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I_Na) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7–21 rats express low density I_Na (9.5–15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC₅₀ value of 39.3 nM. The I_Na expression correlated with a 25 mV more depolarized resting membrane potential (RMP) as compared with non-I_Na-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 μM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I_Na-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I_Na expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 μM TTX. Analysis of the I_Na activation/inactivation curves yields an I_Na “window current” at −40±20 mV, implying a persistent I_Na component being active around the NG2 glia RMP of −45 mV. According to the constant-field equation analysis, this active I_Na component leads to a pNa/pK ratio of 0.14 at RMP which is threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I_Na component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.     

Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan

Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2‐glia. NG2‐glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2‐glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2‐glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2‐glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti‐NG2 antibody in combination with secondary saporin‐conjugated antibody selectively kills NG2‐expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2‐glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2‐glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.     

Transverse relaxation in rat optic nerve

In vitro investigations were performed to study the proton T(2) relaxation spectrum of rat optic nerve. Studies in which the nerve was incubated in a D(2)O-based solution revealed that >98% of the spectrum originated from the water protons. The spectrum was found to consist of three components having relaxation times and sizes similar to those reported in the literature for peripheral nerve. Procedures were taken to confirm the existence of a third optic nerve component and that it was not an artifact of the long-lived water protons of the in vitro incubation solution. Evidence using paramagnetic agents in the incubation solution, which removed the two longest-lived nerve components from the spectrum, revealed the existence of a small fourth component (<10% of total) having a T(2) relaxation time similar to that of the intermediate-lived nerve component. Bathing the nerves in a 10 mM glutamate solution, glutamate known to result in cellular swelling in mammalian central nervous system (CNS), was found to increase the component size of the longest-lived nerve component, suggestive that this component may result from cellular water.     

霍乱毒素及外周神经对视神经远端切断后视网膜谷氨酸能节细胞再生的作用

目的：探讨霍乱毒素(CTx)及外周神经对成年金黄地鼠远端视神经受损后视网膜谷氨酸((Glu)能节细胞(RGCs)再生的作用。方法：远端切断视神经并缝接自体坐骨神经(AG),玻璃体内注射CTx及／或植入小段坐骨神经分支(SN)。动物分为AG CTx组;AG SN组;AG SN CTx组,分别存活4W、5W,荧光金和免疫荧光组织化学双标法标记再生的RGCs。结果：术后5W,AG CTx组;AG SN组;AG SN CTx组Glu免疫反应阳性RGCs再生数分别占再生总数的4.25％、2.50％及6.00％,AG SN组与AG SN CTx组间差异显著。结论：CTx与SN能协同促进视神经远端切断后Glu能节细胞的再生．     

Functional roles of CSPG4/NG2 in chondrosarcoma

CSPG4/NG2 is a multifunctional transmembrane protein with limited distribution in adult tissues including articular cartilage. The purpose of this study was to investigate the possible roles of CSPG4/NG2 in chondrosarcomas and to establish whether this molecule may have potential for targeted therapy. Stable knock‐down of CSPG4/NG2 in the JJ012 chondrosarcoma cell line by shRNA resulted in decreased cell proliferation and migration as well as a decrease in gene expression of the MMP (matrix metalloproteinase) 3 protease and ADAMTS4 (aggrecanase). Chondrosarcoma cells in which CSPG4/NG2 was knocked down were more sensitive to doxorubicin than wild‐type cells. The results indicate that CSPG4/NG2 has roles in regulating chondrosarcoma cell function in relation to growth, spread and resistance to chemotherapy and that anti‐CSPG4/NG2 therapies may have potential in the treatment of surgically unresectable chondrosarcoma.     

大鼠脊髓硫酸软骨素蛋白多糖4细胞的生后发育特征

 目的 研究大鼠脊髓中硫酸软骨素蛋白多糖4（NG2）细胞生后发育特征。方法 采用免疫组织化学及Western blot,观察生后不同发育阶段的脊髓中NG2细胞形态变化。结果 免疫组织化学染色显示,在大鼠生后各发育阶段的脊髓内均有NG2细胞表达;脊髓内NG2细胞胞体逐渐增大,其形态由圆形或卵圆形变为不规则形,突起数目和长度随之增加。与同时期大脑皮层的NG2细胞形态比较,脊髓内的NG2细胞胞体要大,突起短而少。Western blot显示,P1d,脊髓内NG2表达水平最高, P7d开始下降,P21d最低,P60d回升。结论 在大鼠不同发育阶段的脊髓内,NG2细胞形态数量不同, P7d到P21d是脊髓内神经纤维髓鞘主要形成期;脊髓内NG2细胞可能处于少突胶质细胞系发育的早期阶段。     

Effects of electroacupuncture with different frequencies on spinal ionotropic glutamate receptor expression in complete Freund's adjuvant-injected rat

We investigated expressional changes of spinal glutamate receptors by electroacupuncture (EA) in an inflammatory animal model. Inflammation was induced by an intraplantar injection of complete Freund's adjuvant (CFA) into the hindpaw of male Sprague-Dawley rats. Bilateral EA stimulation at 2, 15 and 120 Hz was applied at those acupoints corresponding to Zusanli and Sanyinjiao in man using needles with 3-day intervals for 30 days. Paw edema and mechanical thresholds were measured by a water displacement plethysmometer and Analgesy-Meter, respectively. Edema and mechanical sensitivity of the hindpaw induced by CFA-injection were strongly inhibited by EA stimulation. At 30 days after CFA-injection, effects of EA on ionotropic glutamate receptor (NR-1, NR-2A, GluR-1 and GluR-2/3) expression in association with c-fos and calcitonin gene-related peptide (CGRP) expression were observed in the dorsal horn of the spinal cord using immunohistochemistry. The number of c-fos-like immunostained cells was decreased significantly in the superficial laminae of the dorsal horn by 2Hz EA, but CGRP expression also showed a marked decrease in the same region using the other types of EA stimulation. N-methyl-D-aspartate receptor (NR-1 and NR-2A) expression was attenuated in all regions of the dorsal horn by all types of EA. Of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (GluR-1 and -2/3), only GluR-1 expression was prevented by EA treatment in the superficial laminae and the neck of the dorsal horn. It is concluded that EA treatment can attenuate inflammatory edema and mechanical thresholds in CFA-injected rats through modulating expression of ionotropic glutamate receptors, and especially N-methyl-D-aspartate receptors, in the dorsal horn of the spinal cord.     

Chronic corticosterone administration down-regulates metabotropic glutamate receptor 5 protein expression in the rat hippocampus

Several lines of evidence suggest a dysfunctional glutamate system in major depressive disorder (MDD). Recently, we reported reduced levels of metabotropic glutamate receptor subtype 5 (mGluR5) in postmortem brains in MDD, however the neurobiological mechanisms that induce these abnormalities are unclear. In the present study, we examined the effect of chronic corticosterone (CORT) administration on the expression of mGluR5 protein and mRNA in the rat frontal cortex and hippocampus. Rats were injected with CORT (40 mg/kg s.c.) or vehicled once daily for 21 days. The expression of mGluR5 protein and mRNA was assessed by Western blotting and quantitative real-time PCR (qPCR). In addition, mGluR1 protein was measured in the same animals. The results revealed that while there was a significant reduction (−27%, P=0.0006) in mGluR5 protein expression in the hippocampus from CORT treated rats, mRNA levels were unchanged. Also unchanged were mGluR5 mRNA and protein levels in the frontal cortex and mGluR1 protein levels in both brain regions. Our findings provide the first evidence that chronic CORT exposure regulates the expression of mGluR5 and are in line with previous postmortem and imaging studies showing reduced mGluR5 in MDD. Our findings suggest that elevated levels of glucocorticoids may contribute to impairments in glutamate neurotransmission in MDD.     

Minocycline prevents impaired glial glutamate uptake in the spinal sensory synapses of neuropathic rats

Activation of glutamate receptors and glial cells in the spinal dorsal horn are two fundamental processes involved in the pathogenesis of various pain conditions, including neuropathic pain induced by injury to the peripheral or central nervous systems. Numerous studies have demonstrated that minocycline treatment attenuates allodynic and hyperalgesic behaviors induced by tissue inflammation or nerve injury. However, the synaptic mechanisms by which minocycline prevents hyperalgesia are not fully understood. We recently reported that deficient glutamate uptake by glial glutamate transporters (GTs) is key for the enhanced activation of N-methyl-d-aspartate (NMDA) receptors in the spinal sensory synapses of rats receiving partial sciatic nerve ligation (pSNL). In this study, we investigated how minocycline affects activation of NMDA receptors in the spinal sensory synapses in rats with pSNL by whole cell recordings of NMDA currents in spinal laminea I and II neurons from spinal slices. The effects of minocycline treatments on the dorsal horn expression of glial GTs and astrocyte marker glial fibrillary acidic protein (GFAP) were analyzed by immunohistochemistry. We demonstrated that normalized activation of NMDA receptors in synapses activated by both weak and strong peripheral input in the spinal dorsal horn is temporally associated with attenuated mechanical allodynia in rats with pSNL receiving intraperitoneal injection of minocycline. Minocycline ameliorated both the downregulation of glial GT expression and the activation of astrocytes induced by pSNL in the spinal dorsal horn. We further revealed that preventing deficient glial glutamate uptake at the synapse is crucial for preserving the normalized activation of NMDA receptors in the spinal sensory synapses in pSNL rats treated with minocycline. Our studies suggest that glial GTs may be a potential target for the development of analgesics.     

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.     

大鼠脊髓内囊泡膜谷氨酸转运体1和囊泡膜谷氨酸转运体2阳性纤维和终末在生后发育中的分布和表达变化

目的 探讨囊泡膜谷氨酸转运体1（VGLUT1）和VGLUT2阳性纤维和终末在生后第0天（P0）至第22天（P22）大鼠脊髓内的分布情况和表达变化。 方法 对生后发育P0～P22大鼠的颈膨大和腰膨大部位,进行VGLUT1和VGLUT2免疫组织化学染色。 结果 P0～P22大鼠颈膨大和腰膨大脊髓内均可观察到VGLUT1和VGLUT2阳性纤维和终末,但未观察到胞体样结构。VGLUT1和VGLUT2阳性纤维和终末的分布呈现明显的互补分布,尤其是以脊髓后角更加明显。其中,VGLUT1阳性纤维和终末在P0主要见于颈膨大和腰膨大脊髓后角Ⅲ～Ⅴ层,中间部和前角很微弱。脊髓发育至P3,不仅Ⅲ~Ⅴ层VGLUT1的表达进一步增强,且向外侧部扩展,并在后角基底部Ⅵ层和前角的外侧部（Ⅸ层）也可观察到较强的VGLUT1阳性纤维,呈现一条明显由背内向腹外的带状分布趋势。P7时此带状分布更加明显,并随着发育逐渐向内、外扩展,至P22时已广泛分布于除Ⅱ层之外的整个脊髓。而VGLUT2阳性纤维和终末在P0时即密集出现于脊髓后角Ⅰ～Ⅱ层以及前角的外侧边缘区域;之后随着发育,VGLUT2阳性纤维和终末的分布模式并未发生明显改变,但其密度逐渐有所增加,特别是Ⅰ～Ⅱ层内VGLUT2阳性产物的表达尤为明显。另外,在脊髓白质后索内可见VGLUT1阳性皮质脊髓后束纤维由颈髓（P3）逐渐下降至腰髓（P7）的发育过程。 结论 VGLUT1和VGLUT2阳性纤维和终末在脊髓发育过程中呈现明显不同,且表现出互补分布的特点,这对于进一步理解VGLUT1和VGLUT2在脊髓生后发育过程中不同功能特点可能有意义。     

Specialization of astrocytic membrane at glia limitans in rat optic nerve: freeze-fracture observations

The ultrastructure of astrocytic foot processes at the glia limitans in rat optic nerve was studied by freeze-fracture. Two classes of astrocytic end-foot processes were observed. Most astrocytic foot processes in this region show a high density of orthogonal arrays of P-face particles (assemblies) that are randomly oriented. However, astrocytic foot processes with highly organized columns of assemblies are also observed. These columns are 1-4 assemblies wide (approximately 0.1 micron), with the center-to-center distance between the columns being relatively constant (approximately 0.17 micron). Columns of at least 4 micron length have been observed. The regional specialization of the astrocyte membrane and the differentiation of subpial astrocytes into two structural classes may have important functional implications.     

Instructive niches: environmental instructions that confound NG2 proteoglycan expression and the fate-restriction of CNS progenitors

Cellullar deficits are replenished within the central nervous system (CNS) by progenitors to maintain integrity and recover function after injury. NG2 proteoglycan-expressing progenitors replenish oligodendrocyte populations, but the nature of NG2 proteoglycan may not indicate a restricted population of progenitors. After injury, restorative spatiotemporal cues have the potential ability to regulate divergent fate-choices for NG2 progenitors, and NG2 progenitors are known to produce multiple cell types in vitro. Recent data suggest that NG2 expression is attenuated while protein levels remain high within injurious tissue; thus, NG2 expression is not static but transiently controlled in response to a dynamic interplay of environmental cues. Therefore, NG2 proteoglycan expression could label newly generated cells or be inherited by resident cell populations that produce oligodendrocytes for remyelination, astrocytes that provide trophic support and other cells that contribute to CNS function.     

The effects of development of a food-related operant reflex on the receptor binding of glutamate in the rat brain

Receptor binding of glutamate was studied in the striatum, hippocampus, and cerebral cortex of rats with different abilities to acquire an operant food-related reflex in a Skinner box. The striatum of rapidly-learning rats and rats unable to learn showed significantly higher levels of glutamate binding than controls were not trained in the Skinner box (p<0.05). Striatal receptor binding of glutamate in slow-learning rats was lower than that in rapidly-learning rats and rats which were unable to learn (p<0.05). In the hippocampus, all groups of rats (rapidly-learning, slow-learning, and those unable to learn) showed increased receptor binding of glutamate as compared with controls (p<0.05), in the cerebral cortex, there was a significant decrease in glutamate binding as compared with controls in all groups of animals subjected to training (p<0.05). Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 84, No. 9, pp. 913–919, September, 1998.     

NG2/HMPG modulation of human articular chondrocyte adhesion to type VI collagen is lost in osteoarthritis.

NG2/human melanoma proteoglycan (HMPG) is a chondroitin sulphate proteoglycan (CSPG), expressed by chondrocytes in fetal and in normal and osteoarthritic (OA) adult articular cartilage. NG2/HMPG is a receptor for extracellular matrix proteins, including type VI collagen, and regulates beta1 integrin binding to fibronectin. This study was undertaken to identify whether NG2/HMPG had similar activities in human articular chondrocytes (HACs). Normal and OA adult HAC adhesion to fibronectin, type II or type VI collagen was assessed using a methylene blue assay. The requirement for integrins, NG2/HMPG, and integrin-associated signalling molecules was investigated using anti-beta1 integrin and anti-HMPG antibodies and pharmacological inhibitors of signalling molecules. The adhesion of normal and OA HACs to fibronectin, type II and type VI collagen was beta1 integrin-dependent. Normal HAC adhesion to type VI collagen was stimulated by anti-HMPG antibodies. This effect was inhibited by pertussis toxin. Anti-HMPG antibodies had no effect on OA chondrocyte adhesion to type VI collagen, or on normal and OA cell adhesion to fibronectin and type II collagen. The results show that NG2/HMPG modulates integrin-mediated interactions of normal HACs with type VI collagen. Loss of this activity may be of importance in the progression of osteoarthritis.     

视神经再生机制研究与进展

背景:视神经是广泛用于研究促进或抑制中枢神经系统中轴突再生因素的主要载体之一.目的:对视神经再生机制研究进展予以综述.方法:以Optic nerve,Axon regeneration,Retinal ganglion cell为英文检索词,以视神经,轴突再生,视网膜节细胞为中文检索词,检索发表在PubMed、...     

大鼠视神经吸断伤后相关分子表达的变化

目的：研究视神经损伤后神经胶质细胞去分化的程度及相关分子的表达。方法：成年雄性大鼠视神经吸断伤后,采用免疫组织化学、原位杂交组织化学结合计算机图像分析,检测视神经巢蛋白、胶质纤维酸性蛋白 (GFAP)、髓鞘碱性蛋白(MBP)、神经纤维丝(NF)以及 Nogo-A mRNA 在伤后3、7、14和28 d 4个不同时相点的表达。结果：视神经伤后,巢蛋白表达上调,在28 d 时表达最高;GFAP 表达先下调,7 d 时最低,随后逐渐上调; MBP 表达上调,呈先上升后降低的趋势;NF 表达呈明显下降趋势;Nogo-A mRNA 表达呈上升趋势,3 d 到7 d 的变化最显著。结论：视神经损伤后相关分子表达提示其神经胶质细胞可去分化为神经前体细胞或神经干细胞,但这些前体细胞或干细胞呈不利于神经再生的状态。