中华蜜蜂视觉系统中神经胶质的组成和胚后发育

神经胶质作为视觉系统的重要成分之一, 对视觉系统的发育及功能起着重要的作用。本研究通过组织解剖观察、 免疫组织化学等技术, 对中华蜜蜂Apis cerana cerana幼虫和蛹的视觉系统中神经胶质的类型和发育过程进行了比较研究。研究表明： 在中华蜜蜂视觉系统中, 根据神经胶质的位置和形态主要分为表面神经胶质、 皮层神经胶质和神经纤维网神经胶质3种类型; 神经胶质主要来源于视柄和视叶中的神经胶质前体中心; 神经胶质细胞数量的增加一方面来自于细胞的迁移, 另一方面来自于神经胶质细胞自身的分裂增殖。本研究为昆虫神经胶质的发育以及功能研究提供理论基础。     

Development of glia and blood vessels in the internal capsule of rats

We have explored two aspects of internal capsule development that have not been described previously, namely, the development of glia and of blood vessels. To these ends, we used antibodies to glial fibrillary acidic protein (GFAP) and to vimentin (to identify astrocytes and to radial glia) and Griffonia simplicifolia (lectin; to identify microglia and blood vessels). Further, we made intracardiac injections of Evans Blue to examine the permeability of this dye in the vessels of the internal capsule during neonatal development. Our results show that large numbers of radial glia, astrocytes and microglia are not labelled with these markers in the white matter of the internal capsule until about birth; very few are labelled earlier, during the critical stages of corticofugal and corticopetal axonal ingrowth (E15–E20). The large glial labelling in the internal capsule at birth is accompanied by a dense vascular innervation of the capsule; as with the glia, very few labelled patent vessels are seen earlier. After intracardiac injections of Evans Blue, we find that the blood vessels of the internal capsule are not particularly permeable to Evans Blue. At each age examined (P0, P5, P15), blood vessels are outlined very clearly and there is no diffuse haze of fluorescence within the extracellular space, which is indicative of a leaky vessel. There are three striking differences between the glial environment of the internal capsule and that of the adjacent thalamus. First, the internal capsule is never rich with radial glial fibres (vimentin- and GFAP-immunoreactive) during development (except at P0), whereas the thalamus has many radial fibres from very early development (E15–E17). Second, astrocytes (vimentin- and GFAP-immunoreactive) first become apparent in the internal capsule (E20–P0) well before they do in the thalamus (P15). Third, the internal capsule houses a large transient population of amoeboid microglia (P0–P22), whereas the thalamus does not; only ramified microglia are seen in the thalamus. In summary, our results indicate that all three types of glia in the internal capsule are associated closely with the vasculature, suggesting they may play a role in the development of the blood–brain barrier among the vessels in this white matter region of the forebrain.     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

Glial Cells in Abdominal Ganglia of Crayfish

Glial cells of abdominal ganglia of crayfish have been studied by transmission electron microscopy. Four cell types can be defined: (1) perivascular glial cells, close to the vascular spaces; (2) perineuronal glial cells, the processes of which ensheathe neuron perikarya; (3) adaxonal glial cells ensheathing axons; (4) neuropilar glial cells, associated with synapsing terminals in the neuropile. Neuropilar glia, adaxonal glia and the system formed by perineuronal and perivascular glia separate different functional zones of the neurons from the hemolymph or the electron dense extracellular matrix. These glial arrangements could play a similar role in hemato-neuronal transport. Gap-like junctions between glia and neuron cell bodies are frequent and could be involved in direct triggering of glial activities related to neurons.     

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila central nervous system (CNS) glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. We show that the genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. We demonstrate that the SPGs share direct sibling relationships with neurones and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, we show that Notch signalling positively regulates glial cells missing (gcm) expression in the context of SPG development.     

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.     

Probing the enigma: unraveling glial cell biology in invertebrates

Despite their predominance in the nervous system, the precise ways in which glial cells develop and contribute to overall neural function remain poorly defined in any organism. Investigations in simple model organisms have identified remarkable morphological, molecular, and functional similarities between invertebrate and vertebrate glial subtypes. Invertebrates like Drosophila and Caenorhabditis elegans offer an abundance of tools for in vivo genetic manipulation of single cells or whole populations of glia, ease of access to neural tissues throughout development, and the opportunity for forward genetic analysis of fundamental aspects of glial cell biology. These features suggest that invertebrate model systems have high potential for vastly improving the understanding of glial biology. This review highlights recent work in Drosophila and other invertebrates that reveal new insights into basic mechanisms involved in glial development.     

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.     

RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia

Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized. To gain an understanding of how glia migrate, we specifically labeled the peripheral glia of Drosophila melanogaster using an Actin-GFP marker and analyzed their development in the embryonic PNS. It was found that Actin cytoskeleton is dynamically rearranged during glial cell migration. The peripheral glia were observed to migrate as a continuous chain of cells, with the leading glial cells appearing to participate to the greatest extent in exploring the extracellular surroundings with filopodia-like Actin containing projections. We hypothesized that the small GTPases Rho, Rac and Cdc42 are involved in Actin cytoskeletal rearrangements that underlie peripheral glial migration and nerve ensheathement. To test this, transgenic forms of the GTPases were ectopically expressed specifically in the peripheral glia during their migration and wrapping phases. The effects on glial Actin-GFP distribution and the overall effects on glial cell migration and morphological development were assessed. We found that RhoA and Rac1 have distinct roles in peripheral glial cell migration and nerve ensheathement; however, Cdc42 does not have a significant role in peripheral glial development. RhoA and Rac1 gain-of-function and loss-of-function mutants had both disruption of glial cell development and secondary effects on sensory axon fasciculation. Together, Actin cytoskeletal dynamics is an integral part of peripheral glial migration and nerve ensheathement, and is mediated by RhoA and Rac1.     

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β‐catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co‐cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt‐dependent target proteins that may mediate the axonal growth.     

Glial Contributions to Neural Function and Disease

The nervous system consists of neurons and glial cells. Neurons generate and propagate electrical and chemical signals, whereas glia function mainly to modulate neuron function and signaling. Just as there are many different kinds of neurons with different roles, there are also many types of glia that perform diverse functions. For example, glia make myelin; modulate synapse formation, function, and elimination; regulate blood flow and metabolism; and maintain ionic and water homeostasis to name only a few. Although proteomic approaches have been used extensively to understand neurons, the same cannot be said for glia. Importantly, like neurons, glial cells have unique protein compositions that reflect their diverse functions, and these compositions can change depending on activity or disease. Here, I discuss the major classes and functions of glial cells in the central and peripheral nervous systems. I describe proteomic approaches that have been used to investigate glial cell function and composition and the experimental limitations faced by investigators working with glia.The nervous system is composed of neurons and glial cells that function together to create complex behaviors. Traditionally, glia have been considered to be merely passive contributors to brain function, resulting in a pronounced neurocentric bias among neuroscientists. Some of this bias reflects a paucity of knowledge and tools available to study glia. However, this view is rapidly changing as new tools, model systems (culture and genetic), and technologies have permitted investigators to show that glia actively sculpt and modulate neuronal properties and functions in many ways. Glia have been thought to outnumber neurons by 10:1, although more recent studies suggest the ratio in the human brain is closer to 1:1 with region-specific differences (1). There are many different types of glia, some of which are specific to the central nervous system (CNS),¹ whereas others are found only in the peripheral nervous system (PNS). The main types of CNS glia include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia. In the PNS, the main glial cells are Schwann cells, satellite cells, and enteric glia. These cells differ and are classified according to their morphologies, distinct anatomical locations in the nervous system, functions, developmental origins, and unique molecular compositions. Among the different classes of glia there are additional subclasses that reflect further degrees of specialization. In this review, I will discuss the characteristics and functions of the major glial cell types including astrocytes, microglia, and the myelin-forming oligodendrocytes (CNS) and Schwann cells (PNS). Because of space limitations, it is impossible to give a complete accounting of all glia and what is known about each of these cell types. Therefore, I encourage the interested reader to refer to some of the many excellent reviews referenced below that focus on individual glial cell types. Finally, I will discuss proteomic studies of glial cell function and some of the unique challenges investigators face when working with these cells.     

Generation and early differentiation of glial cells in the first optic ganglion of Drosophila melanogaster.

We have examined the generation and development of glial cells in the first optic ganglion, the lamina, of Drosophila melanogaster. Previous work has shown that the growth of retinal axons into the developing optic lobes induces the terminal cell divisions that generate the lamina monopolar neurons. We investigated whether photoreceptor ingrowth also influences the development of lamina glial cells, using P element enhancer trap lines, genetic mosaics and birthdating analysis. Enhancer trap lines that mark the differentiating lamina glial cells were found to require retinal innervation for expression. In mutants with only a few photoreceptors, only the few glial cells near ingrowing axons expressed the marker. Genetic mosaic analysis indicates that the lamina neurons and glial cells are readily separable, suggesting that these are derived from distinct lineages. Additionally, BrdU pulse-chase experiments showed that the cell divisions that produce lamina glia, unlike those producing lamina neurons, are not spatially or temporally correlated with the retinal axon ingrowth. Finally, in mutants lacking photoreceptors, cell divisions in the glial lineage appeared normal. We conclude that the lamina glial cells derive from a lineage that is distinct from that of the L-neurons, that glia are generated independently of photoreceptor input, and that completion of the terminal glial differentiation program depends, directly or indirectly, on an inductive signal from photoreceptor axons.     

Two forms of cerebellar glial cells interact differently with neurons in vitro

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.     

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal–glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10–P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-βIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and βIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and βIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal–glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.     

Evolutionarily conserved concepts in glial cell biology

The evolutionary conservation of glial cells has been appreciated since Ramon y Cajal and Del Rio Hortega first described the morphological features of cells in the nervous system. We now appreciate that glial cells have essential roles throughout life in most nervous systems. The field of glial cell biology has grown exponentially in the last ten years. This new wealth of knowledge has been aided by seminal findings in non-mammalian model systems. Ultimately, such concepts help us to understand glia in mammalian nervous systems. Rather than summarizing the field of glial biology, I will first briefly introduce glia in non-mammalian models systems. Then, highlight seminal findings across the glial field that utilized non-mammalian model systems to advance our understanding of the mammalian nervous system. Finally, I will call attention to some recent findings that introduce new questions about glial cell biology that will be investigated for years to come.