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The problem of astrocyte identity
Abstract
Astrocytes were the original neuroglia of Ramón y Cajal but after 100 years there is no satisfactory definition of what should comprise this class of cells. This essay takes a historical and philosophical approach to the question of astrocytic identity. The classic approach of identification by morphology and location are too limited to determine new members of the astrocyte population. I also critically evaluate the use of protein markers measured by immunoreactivity, as well as the newer technique of marking living cells by using promoters for these same proteins to drive reporter genes. These two latter approaches have yielded an expanded population of astrocytes with diverse functions, but also mark cells that traditionally would not be defined as astrocytes. Thus we need a combination of measures to define an astrocyte but it is not clear what this combination should be. The molecular approach, especially promoter driven fluorescent reporter genes, does have the advantage of pre marking living astrocytes for electrophysiological or imaging recordings. However, lack of sufficient understanding of the behavior of the inserted constructs has led to unclear results. This approach will no doubt be perfected with time but at present an acceptable, practical definition of what constitutes the class of astrocytes remains elusive.
... In addition, they may have different gene expression characteristics and are considered more active than mature adult brain cells [37]. The experimental results from neonatal cells cannot be directly transferred to the adult cells [27,30,32]. Therefore, adult brain-derived neuroglial cells provide a valuable and convenient model for experiments, as their pathophysiological mechanisms cannot be equally studied in neonatal culture [27,30,32,33]. ...
... The experimental results from neonatal cells cannot be directly transferred to the adult cells [27,30,32]. Therefore, adult brain-derived neuroglial cells provide a valuable and convenient model for experiments, as their pathophysiological mechanisms cannot be equally studied in neonatal culture [27,30,32,33]. ...
... Experimental results from neonatal cells cannot be directly extrapolated to adults, highlighting the superiority of adult astrocyte and oligodendrocyte cultures in these cases. Factors that may influence brain cell isolation and increase case-to-case variability include age differences in neonatal donors and different donor tissue conditions [27,30,[32][33][34]. In addition, the tissue source for adult astrocyte isolation is much more readily available than neonatal brain tissue obtained during elective abortions at 9 to 22 weeks of age [32,38,39]. ...
In recent decades, cell biology has made rapid progress. Cell isolation and cultivation techniques, supported by modern laboratory procedures and experimental capabilities, provide a wide range of opportunities for in vitro research to study physiological and pathophysiological processes in health and disease. They can also be used very efficiently for the analysis of biomaterials. Before a new biomaterial is ready for implantation into tissues and widespread use in clinical practice, it must be extensively tested. Experimental cell models, which are a suitable testing ground and the first line of empirical exploration of new biomaterials, must contain suitable cells that form the basis of biomaterial testing. To isolate a stable and suitable cell culture, many steps are required. The first and one of the most important steps is the collection of donor tissue, usually during a surgical procedure. Thus, the collection is the foundation for the success of cell isolation. This article explains the sources and neurosurgical procedures for obtaining brain tissue samples for cell isolation techniques, which are essential for biomaterial testing procedures.
... Consequently, there are two significant challenges when considering astrocytes as biomaterials for brain research: (I) maintaining a non-activated state of astrocytes, with low GFAP expression; and (II) achieving a physiological morphology of the cells in culture. GFAP is a prototypical marker for the immunocytochemical identification of astrocytes because it is a reliable and sensitive marker [21,[44][45][46]. Moreover, it is also crucial in biomaterial research and development of astrocyte cell models. ...
... On the contrary, if the biomaterial increases GFAP expression, this implies a more reactive astrocyte phenotype that may not have a positive effect on regeneration [21,47]. This is important for biomaterial development because novel biomaterials need to stimulate astrocytes towards the phenotype that promotes axonal regeneration and neuronal survival [22,45]. ...
... A physiological morphology of astrocytes can be achieved by culturing the cells in a three-dimensional matrix that provides structural support and appropriate extracellular matrix factors, which allows the formation of the characteristic star-shaped morphology and low GFAP expression, which is an indicator of astrocyte activation [22,45,46]. ...
The development of in vitro neural tissue analogs is of great interest for many biomedical engineering applications, including the tissue engineering of neural interfaces, treatment of neurodegenerative diseases, and in vitro evaluation of cell–material interactions. Since astrocytes play a crucial role in the regenerative processes of the central nervous system, the development of biomaterials that interact favorably with astrocytes is of great research interest. The sources of human astrocytes, suitable natural biomaterials, guidance scaffolds, and ligand patterned surfaces are discussed in the article. New findings in this field are essential for the future treatment of spinal cord and brain injuries.
... An increased or decreased number of positive cells can be observed following injury, as well as a change in the intensity of expression of the respective marker. At the same time, cell markers are suitable for detailed study of the morphology of individual cell types, especially in neural tissue, here they exhibit a pronounced diversity of their structure [63][64][65][66][67][68][69]. Figure 2 displays cellular markers used for the morphological categorization of astrocytes. ...
... An increased or decreased number of positive cells can be observed following injury, as well as a change in the intensity of expression of the respective marker. At the same time, cell markers are suitable for detailed study of the morphology of individual cell types, especially in neural tissue, here they exhibit a pronounced diversity of their structure [63][64][65][66][67][68][69]. Figure 2 displays cellular markers used for the morphological categorization of astrocytes. Proteins specific to astrocytes include vimentin, desmin, synemin, and glial fibrillary acidic protein (GFAP). ...
There is a growing interest in glial cells in the central nervous system due to their important role in maintaining brain homeostasis under physiological conditions and after injury. A significant amount of evidence has been accumulated regarding their capacity to exert either pro-inflammatory or anti-inflammatory effects under different pathological conditions. In combination with their proliferative potential, they contribute not only to the limitation of brain damage and tissue remodeling but also to neuronal repair and synaptic recovery. Moreover, reactive glial cells can modulate the processes of neurogenesis, neuronal differentiation, and migration of neurons in the existing neural circuits in the adult brain. By discovering precise signals within specific niches, the regulation of sequential processes in adult neurogenesis holds the potential to unlock strategies that can stimulate the generation of functional neurons, whether in response to injury or as a means of addressing degenerative neurological conditions. Cerebral ischemic stroke, a condition falling within the realm of acute vascular disorders affecting the circulation in the brain, stands as a prominent global cause of disability and mortality. Extensive investigations into glial plasticity and their intricate interactions with other cells in the central nervous system have predominantly relied on studies conducted on experimental animals, including rodents and primates. However, valuable insights have also been gleaned from in vivo studies involving poststroke patients, utilizing highly specialized imaging techniques. Following the attempts to map brain cells, the role of various transcription factors in modulating gene expression in response to cerebral ischemia is gaining increasing popularity. Although the results obtained thus far remain incomplete and occasionally ambiguous, they serve as a solid foundation for the development of strategies aimed at influencing the recovery process after ischemic brain injury.
... 73,75,76 Second, GFAP immunoreactivity reveals only the primary branches of astrocytes while missing terminal leaflets or small endfeet altogether. 73,75,77,78,79,80,81 Vimentin is another member of F I G U R E 3 Labelling RSA and protoplasmic astrocytes by immunohistochemistry. Confocal micrograph showing staining with three antigens for RSA and/or astrocytes (A1): GFAP (green; A2), S100β (red; A3), and SOX2 (white; A4) in the adult DG (scale bars 20 μm). The schematic diagram shows immunostaining markers for astrocytes, RSA, and progenitors. ...
... 173 Increase in GFAP expression, however, is not an ideal marker for reactive astrocytes since (i) GFAP expression in astrocytes, as mentioned previously, differs between brain regions; (ii) GFAP expression is modulated by various extrinsic and intrinsic factors and often indicates adaptive plasticity. 73,75,78,176,177 Furthermore, an increase in GFAP does not report full cellular morphology, and often increase in GFAPpositive profiles does not coincide with actual cellular hypertrophy. 178 In the brain trauma, stroke, immune, or infectious attacks reactive astrocytes become proliferative. ...
Adult neurogenesis is a striking example of neuroplasticity, which enables adaptive network remodelling in response to all forms of environmental stimulation in physiological and pathological contexts. Dysregulation or cessation of adult neurogenesis contributes to neuropathology negatively affecting brain functions and hampering regeneration of the nervous tissue, while targeting adult neurogenesis may provide the basis for potential therapeutic interventions. Neural stem cells in the adult mammalian brain are at the core and at the entry point of adult neurogenesis. By their origin and properties these cells belong to astroglia, and are represented by stem radial astrocytes (RSA) which exhibit multipotent "stemness". In the neurogenic niches RSA interact with other cellular components, including protoplasmic astrocytes, which in turn regulate their neurogenic activity. In pathology, RSA become reactive, which affects their neurogenic capabilities, whereas reactive parenchymal astrocytes up-regulate stem cell hallmarks and are able to generate progeny that remain within astrocyte lineage. What makes RSA special is their multipotency, represented by self-renewing capacity capability to generate other cellular types as progeny. A broad understanding of the cellular features of RSA and parenchymal astrocytes provides an insight into the machinery that promotes/suppresses adult neurogenesis, clarifying principles of network remodelling. In this review we discuss the cellular hallmarks, research tools and models of RSA and astrocytes of the subventricular zone along the lateral ventricle and dentate gyrus of the hippocampus. We also discuss RSA in ageing, which has great impact on the proliferative capacity of RSA, as well as the potential of RSA and astrocytes in therapeutic strategies aimed at cell replacement and regeneration.
... The gradient of height along the bridges which connects islands extends approximately 10 μm from the plateau characterising the homogeneous film (Figure 1c,d). Astrocytes and their processes often surround CNS blood vessels, synaptic areas [55] or axon tracts [56]. These brain features have similar shapes (i.e., elongated structures) to the bridges in our patterns. ...
... In their natural brain environment, astrocytes encounter and interact with elongated structures of micrometric dimensions, such as blood vessels and neuronal cells [55][56][57][58][59]. We therefore performed experiments with astrocyte cultures on micropatterned substrates with nanotopographical features that mimic such kinds of elongated geometries. ...
Astrocytes’ organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation.
... Fig. 3B). However, this was not surprising since AC expression of GFAP varies in vivo (52). ...
... When ACs transition to a reactive state, it is typically accompanied by an increase in cellular diameter, GFAP expression, and the number of astrocytic processes (52,53). For these reasons, we speculated that the unlabeled nuclei in MVNs could be those of ACs with no GFAP expression. ...
The bulk flow of interstitial fluid through tissue is an important factor in human biology, including the development of brain microvascular networks (MVNs) with the blood-brain barrier (BBB). Bioengineering perfused, functional brain MVNs has great potential for modeling neurovascular diseases and drug delivery. However, most in vitro models of brain MVNs do not implement interstitial flow during the generation of microvessels. Using a microfluidic device (MFD), we cultured primary human brain endothelial cells (BECs), pericytes, and astrocytes within a 3D fibrin matrix with (flow) and without (static) interstitial flow. We found that the bulk flow of interstitial fluid was beneficial for both BEC angiogenesis and vasculogenesis. Brain MVNs cultured under flow conditions achieved anastomosis and were perfusable, whereas those under static conditions lacked connectivity and the ability to be perfused. Compared to static culture, microvessels developed in flow culture exhibited an enhanced vessel area, branch length and diameter, connectivity, and longevity. Although there was no change in pericyte coverage of microvessels, a slight increase in astrocyte coverage was observed under flow conditions. In addition, the immunofluorescence intensity of basal lamina proteins, collagen IV and laminin, was nearly doubled in flow culture. Lastly, the barrier function of brain microvessels was enhanced under flow conditions, as demonstrated by decreased dextran permeability. Taken together, these results highlighted the importance of interstitial flow in the in vitro generation of perfused brain MVNs with characteristics similar to those of the human BBB.
... Most commonly used markers of AS are GFAP and S100β [17][18][19], although not all AS are GFAP positives [20]. Other useful markers include N-myc downstream-regulated gene 2 (NDRG2) [19], caveolin-3 [21], connexin 30 [22], connexin 43 [21] and aldehyde dehydrogenase 1 L1 [23]. ...
Astrocytes (AS) are the most abundant glial cells in the central nervous system (CNS). They have various morphologies and numerous (50-60) branching prolongations, with roles in the maintenance of the CNS function and homeostasis. AS in the optic nerve head (ONH) have specific distribution and function and are involved in the pathogenesis of glaucoma and other neural diseases, modify their morphologies, location, immune phenotype, and ultrastructure, thus being the key players in the active remodeling processes of the ONH.
... Over the past 10 years, scientific research has revealed that the astrocyte [1] possesses much more diversity in its functionality that was commonly thought beyond its subservient role to the neuron [2][3][4]. Such functionality has traversed endocannabinoid synthesis [5], immunological signalling [6][7][8], the production of cytokines and chemokines [9] and the unanticipated modulation of the synaptic cleft between neurons [10][11][12]. ...
The ‘Astrocyte Network’ and the understanding of its communication has been posed as a new grand challenge to be investigated by contemporary science. However, communication studies in astrocyte networks have investigated traditional petri-dish in vitro culture models where cells are closely packed and can deviate from the stellate form observed in the brain. Using novel cell patterning approaches, highly organised, regular grid networks of astrocytes on chip, to single-cell fidelity are constructed, permitting a stellate-like in vitro network model to be realised. By stimulating the central cell with a single UV nanosecond laser pulse, the initiation/propagation pathways of stellate-like networks are re-explored. The authors investigate the mechanisms of intercellular Ca ²⁺ communication and discover that stellate-like networks of adult human astrocytes in vitro actually exploit extracellular ATP release as their dominant propagation pathway to cells in the network locally; being observed even down to the nearest neighbour and next nearest neighbouring cells—contrary to the reported gap junction. This discovery has significant ramifications to many neurological conditions such as epilepsy, stroke and aggressive astrocytomas where gap junctions can be targeted. In cases where such gap junction targeting has failed, this new finding suggests that these conditions should be re-visited and the ATP transmission pathway targeted instead.
... In reality, most of the astrocytes do not have a star-like morphology and many astrocytes do not express GFAP (Kimelberg, 2004); indeed, the normal levels of GFAP expression vary quite considerably between brain regions. For example, GFAP is expressed by virtually every Bergmann glial cell in the cerebellum and by fibrous astrocytes in white matter, whereas only about 15-20 per cent of protoplasmic astrocytes express GFAP in the cortex of mature animals. ...
... Previous studies that focused mainly on the alteration of GFAP immunoreactive astrocytes in the MVN after vestibular loss indicated that astrocytes contributed to the process of VC (Campos-Torres et al., 2005;Tighilet and Chabbert, 2019). However, the challenge is that in the CNS there is a large population of astrocytes with diverse function which are not always GFAP immunoreactive (Kimelberg, 2004). Although an increase of GFAP immunostaining was confirmed in the ipsilateral MVN of mice in our experiments, we suggest other gene markers including Agt, Slc6a11, Gja1, Gjb6, Fgfr3, and Aqp4 to complement the identification classification of the astrocytes. ...
Astrocytes are highly heterogeneous and involved in different aspects of fundamental functions in the central nervous system (CNS). However, whether and how this heterogeneous population of cells reacts to the pathophysiological challenge is not well understood. To investigate the response status of astrocytes in the medial vestibular nucleus (MVN) after vestibular loss, we examined the subtypes of astrocytes in MVN using single-cell sequencing technology in a unilateral labyrinthectomy mouse model. We discovered four subtypes of astrocytes in the MVN with each displaying unique gene expression profiles. After unilateral labyrinthectomy, the proportion of the astrocytic subtypes and their transcriptional features on the ipsilateral side of the MVN differ significantly from those on the contralateral side. With new markers to detect and classify the subtypes of astrocytes in the MVN, our findings implicate potential roles of the adaptive changes of astrocyte subtypes in the early vestibular compensation following peripheral vestibular damage to reverse behavioral deficits.
... The area of astrocytes was determined by outlining individual astrocytes observed as GFAP immunosignal. Because GFAP protein is mostly localized in the major primary and secondary branches [55,56], undetectable terminal astrocyte processes were excluded from this analysis. Comparative analyses were only performed in the case of images taken with the same acquisition parameters. ...
Background:
Epilepsy affects millions of people worldwide, yet we still lack a successful treatment for all epileptic patients. Most of the available drugs modulate neuronal activity. Astrocytes, the most abundant cells in the brain, may constitute alternative drug targets. A robust expansion of astrocytic cell bodies and processes occurs after seizures. Highly expressed in astrocytes, CD44 adhesion protein is upregulated during injury and is suggested to be one of the most important proteins associated with epilepsy. It connects the astrocytic cytoskeleton to hyaluronan in the extracellular matrix, influencing both structural and functional aspects of brain plasticity.
Methods:
Herein, we used transgenic mice with an astrocyte CD44 knockout to evaluate the impact of the hippocampal CD44 absence on the development of epileptogenesis and ultrastructural changes at the tripartite synapse.
Results:
We demonstrated that local, virally-induced CD44 deficiency in hippocampal astrocytes reduces reactive astrogliosis and decreases the progression of kainic acid-induced epileptogenesis. We also observed that CD44 deficiency resulted in structural changes evident in a higher dendritic spine number along with a lower percentage of astrocyte-synapse contacts, and decreased post-synaptic density size in the hippocampal molecular layer of the dentate gyrus.
Conclusions:
Overall, our study indicates that CD44 signaling may be important for astrocytic coverage of synapses in the hippocampus and that alterations of astrocytes translate to functional changes in the pathology of epilepsy.
... 36,58 In more mature healthy brains, only a fraction (w15e20%) of astrocytes can be visualised with GFAP antibodies. 59,60 Immunostaining against GFAP labels only cytoskeletal structures associated with main processes (Fig. 3.6AeC), leaving peripheral leaflets (which contribute to the main portion of cellular arborisation) and small endfeet indiscernible. 61,62 ...
... [3] This article will explore the contrast among the historical remodelings regarding the understanding of astrocyte molecular biology while focusing on the review of avant-garde updates central to astrocyte functioning. It is already well put by Harold K Kimelberg (2004) [4], "Astrocytes had been the initial neuroglia of Ramón y Cajal but even after 100 years there is no adequate interpretation of what should comprise these cells," Surely more systematic examination into this would extract an integrated and thorough understanding of astrocytes but this could borrow more time, perhaps years. This article puts into perspective, the manifold scope of astrocyte biology by producing organized discussions regarding several essential themes such as astrocytes as intermediaries to the regulation of neurodegenerative diseases, neural modulation and discoveries of astrocyte-associated therapeutic targets. ...
Throughout the history of the ascent of cognitive sciences, cells of the robust and rather resourceful bio-entity we call the brain have enjoyed particular attention, deliberation and scrutiny. Among these are the tiles that pile up throughout CNS by outnumbering neurons fivefold, called Astrocytes. (Michael V et al 2009) [1] Astrocytes played a passive role in the learning of neuroscience, however, recent studies have invoked compelling novelty that attends more to their contribution as astrocyte-assisted neural signalling, astrocyte-associated immunological activity (Dong et al 2001) [2] and Ca-based form of excitability (Fiacco TA et al 2008). [3] This article will explore the contrast among the historical remodelings regarding the understanding of astrocyte molecular biology while focusing on the review of avant-garde updates central to astrocyte functioning. It is already well put by Harold K Kimelberg (2004) [4], "Astrocytes had been the initial neuroglia of Ramón y Cajal but even after 100 years there is no adequate interpretation of what should comprise these cells," Surely more systematic examination into this would extract an integrated and thorough understanding of astrocytes but this could borrow more time, perhaps years. This article puts into perspective, the manifold scope of astrocyte biology by producing organized discussions regarding several essential themes such as astrocytes as intermediaries to the regulation of neurodegenerative diseases, neural modulation and discoveries of astrocyte-associated therapeutic targets.
... Astrocytes are star-shaped glial cells with radial processes that play essential functions as blood-brain barrier formation and maintenance (Abbott et al., 2006;Daneman and Prat, 2015;Janzer and Raff, 1987), ionic environment regulation (Anderson and Swanson, 2000;Sattler and Rothstein, 2006;Seifert et al., 2006;Simard and Nedergaard, 2004;Strohschein et al., 2011), control of neurogenesis and glycogen storage (Brown et al., 2005;Brown and Ransom, 2007;Matsui et al., 2017), neurometabolic uncoupling (Magistretti, 2006), iron-induce antioxidant protection (Hoepken et al., 2004;Oide et al., 2006;Regan et al., 2002), among others. When the brain tissue is damaged, astrocytes exhibit hypertrophy (Hol and Pekny, 2015;Kimelberg, 2004), and alter gene expression of glial fibrillary acidic protein (GFAP) resulting in a reactive state (Sofroniew, 2009). Previous reports indicate that astrocytes of aged rats showed an enhanced expression of GFAP and a hypertrophic phenotype, similarly to old humans, and senescence-accelerated animal models (Clarke et al., 2018;Cotrina and Nedergaard, 2002;Hol and Pekny, 2015;Kohama et al., 1995;Nichols et al., 1993;Rodríguez et al., 2014;Rozovsky et al., 1998;Woodruff-Pak, 2008;Wu et al., 2005;Yoshida et al., 1996). ...
Astrocytes perform multiple essential functions in the brain showing morphological changes. Hypertrophic astrocytes are commonly observed in cognitively healthy aged animals, implying a functional defense mechanism without losing neuronal support. In neurodegenerative diseases, astrocytes show morphological alterations, such as decreased process length and reduced number of branch points, known as astroglial atrophy, with detrimental effects on neuronal cells. The common marmoset (Callithrix jacchus) is a non-human primate that, with age, develops several features that resemble neurodegeneration. In this study, we characterize the morphological alterations in astrocytes of adolescent (mean 1.75 y), adult (mean 5.33 y), old (mean 11.25 y), and aged (mean 16.83 y) male marmosets. We observed a significantly reduced arborization in astrocytes of aged marmosets compared to younger animals in the hippocampus and entorhinal cortex. These astrocytes also show oxidative damage to RNA and increased nuclear plaques in the cortex and tau hyperphosphorylation (AT100). Astrocytes lacking S100A10 protein show a more severe atrophy and DNA fragmentation. Our results demonstrate the presence of atrophic astrocytes in the brains of aged marmosets.
... Whereas the majority of (~80-90%) astrocytes in the hippocampal formation do express GFAP, this percent in EC is only at 15-20% [27]. Therefore, in order to address the morphometric changes of astrocytes in the EC, we decided to consider another astrocytic marker and trophic factor: the S100β protein. ...
Astrocytes contribute to the progression of neurodegenerative diseases, including Alzheimer's disease (AD). Here, we report the neuroanatomical and morphometric analysis of astrocytes in the entorhinal cortex (EC) of the aged wild type (WT) and triple transgenic (3xTg-AD) mouse model of AD. Using 3D confocal microscopy, we determined the surface area and volume of positive astrocytic profiles in male mice (WT and 3xTg-AD) from 1 to 18 months of age. We showed that S100β-positive astrocytes were equally distributed throughout the entire EC in both animal types and showed no changes in Nv (number of cells/mm3) nor in their distribution at the different ages studied. These positive astrocytes, demonstrated an age-dependent gradual increase in their surface area and in their volume starting at 3 months of age, in both WT and 3xTg-AD mice. This last group demonstrated a large increase in both surface area and volume at 18 months of age when the burden of pathological hallmarks of AD is present (69.74% to 76.73% in the surface area and the volume, for WT and 3xTg-AD mice respectively). We observed that these changes were due to the enlargement of the cell processes and to less extend the somata. In fact, the volume of the cell body was increased by 35.82% in 18-month-old 3xTg-AD compared to WT. On the other hand, the increase on the astrocytic processes were detected as soon as 9 months of age where we found an increase of surface area and volume (36.56% and 43.73%, respectively) sustained till 18 month of age (93.6% and 113.78%, respectively) when compared age-matched non-Tg mice. Moreover, we demonstrated that these hypertrophic S100β-positive astrocytes were mainly associated with Aβ plaques. Our results show a severe atrophy in GFAP cytoskeleton in all cognitive areas; whilst within the EC astrocytes independent to this atrophy show no changes in GS and S100β; which can play a key role in the memory impairment.
... 62,63 Therefore, it is possible to find abnormal plasma GFAP levels in some participants irrespective of mutation status at an earlier EYO. It is also important to note that while GFAP is a putative astrocyte reactivity marker in the literature,64 not all astrocytes produce GFAP.[65][66][67][68][69][70] Further, given thatPSEN1 mutations before or after codon 200 position are known to affect the balance of parenchymal versus vascular Aβ burden, 71,72 pilot findings on stratifying PSEN1 mutation carriers based on mutation position before or after codon 200 or the mutation type (PSEN1 or APP) showed that plasma GFAP levels are significantly elevated in the following order: PSEN1 MC before codon 200 followed by PSEN1 MC after codon 200 followed by APP MC, compared to non-carriers (Figure S4 in supporting information); however, given the modest sample size of the subgroups, further confirmatory studies are required. ...
Background:
Glial fibrillary acidic protein (GFAP) is a promising candidate blood-based biomarker for Alzheimer's disease (AD) diagnosis and prognostication. The timing of its disease-associated changes, its clinical correlates, and biofluid-type dependency will influence its clinical utility.
Methods:
We evaluated plasma, serum, and cerebrospinal fluid (CSF) GFAP in families with autosomal dominant AD (ADAD), leveraging the predictable age at symptom onset to determine changes by stage of disease.
Results:
Plasma GFAP elevations appear a decade before expected symptom onset, after amyloid beta (Aβ) accumulation and prior to neurodegeneration and cognitive decline. Plasma GFAP distinguished Aβ-positive from Aβ-negative ADAD participants and showed a stronger relationship with Aβ load in asymptomatic than symptomatic ADAD. Higher plasma GFAP was associated with the degree and rate of neurodegeneration and cognitive impairment. Serum GFAP showed similar relationships, but these were less pronounced for CSF GFAP.
Conclusion:
Our findings support a role for plasma GFAP as a clinical biomarker of Aβ-related astrocyte reactivity that is associated with cognitive decline and neurodegeneration.
Highlights:
Plasma glial fibrillary acidic protein (GFAP) elevations appear a decade before expected symptom onset in autosomal dominant Alzheimer's disease (ADAD). Plasma GFAP was associated to amyloid positivity in asymptomatic ADAD. Plasma GFAP increased with clinical severity and predicted disease progression. Plasma and serum GFAP carried similar information in ADAD, while cerebrospinal fluid GFAP did not.
... 62,63 Therefore, it is possible to find abnormal plasma GFAP levels in some participants irrespective of mutation status at an earlier EYO. It is also important to note that while GFAP is a putative astrocyte reactivity marker in the literature,64 not all astrocytes produce GFAP.[65][66][67][68][69][70] Further, given thatPSEN1 mutations before or after codon 200 position are known to affect the balance of parenchymal versus vascular Aβ burden, 71,72 pilot findings on stratifying PSEN1 mutation carriers based on mutation position before or after codon 200 or the mutation type (PSEN1 or APP) showed that plasma GFAP levels are significantly elevated in the following order: PSEN1 MC before codon 200 followed by PSEN1 MC after codon 200 followed by APP MC, compared to non-carriers (Figure S4 in supporting information); however, given the modest sample size of the subgroups, further confirmatory studies are required. ...
Glial fibrillary acidic protein (GFAP) is a promising candidate blood-based biomarker for Alzheimer’s disease (AD) diagnosis and prognostication. The timing of its disease-associated changes, its clinical correlates, and biofluid-type dependency will influence its clinical utility. We evaluated plasma, serum, and CSF GFAP in families with autosomal dominant AD (ADAD), leveraging the predictable age at symptom onset to determine changes by stage of disease. Plasma GFAP elevations appear a decade before expected symptom onset, after β-amyloid accumulation and prior to neurodegeneration and cognitive decline. Plasma GFAP distinguished β-amyloid-positive from β-amyloid-negative ADAD participants and showed a stronger relationship with β-amyloid load in asymptomatic than symptomatic ADAD. Higher plasma GFAP was associated with the degree and rate of neurodegeneration and cognitive impairment. Serum GFAP showed similar relationships, but these were less pronounced for CSF GFAP. Our findings support a role for plasma GFAP as a clinical biomarker for β-amyloid-associated cognitive deterioration in AD.
... Ultimately, the mechanisms underlying the elaboration of astrocytic processes remain to be fully elucidated, but a growing list of the literature has shed light on the potential role of actin dynamics. The major processes of astrocytes are rich in intermediate filaments such as glia fibrillary acidic protein (GFAP), a protein frequently used as a marker to identify astrocytes [5,23], and microtubule proteins [24,25] such as atubulin [26]. Both microtubules and intermediate filaments are restricted to the primary and secondary branches that emanate from the soma [25]. ...
Astrocytes represent an abundant type of glial cell involved in nearly every aspect of central nervous system (CNS) function, including synapse formation and maturation, ion and neurotransmitter homeostasis, blood–brain barrier maintenance, as well as neuronal metabolic support. These various functions are enabled by the morphological complexity that astrocytes adopt. Recent experimental advances in genetic and viral labeling, lineage tracing, and live- and ultrastructural imaging of miniscule astrocytic sub-compartments reveal a complex morphological heterogeneity that is based on the origin, local function, and environmental context in which astrocytes reside. In this minireview, we highlight recent findings that reveal the plastic nature of astrocytes in the healthy brain, particularly at the synapse, and emerging technologies that have advanced our understanding of these morphologically complex cells.
... Astrocytes have been historically classified based on their morphology, anatomical location, and marker gene expression (Barres, 2008;Sofroniew & Vinters, 2010). Indeed, canonical astrocyte markers do not always label the entire population of astrocytes and/or the same embedded circuitries (Emsley & Macklis, 2006;Kimelberg, 2004;Preston et al., 2019). Recent studies based on bulk and single-cell (sc) RNA sequencing (Seq) analyses have revealed that astrocytes exhibit a high molecular diversity within the same, as well as across different brain regions (Ben Haim & Rowitch, 2017;Boisvert et al., 2018;Morel et al., 2017). ...
Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nu-cleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following the expo-sure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile de-pendent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates time- and cell- specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pat-tern compared to other cell types. Lastly, we topographically and molecularly characterized as-trocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family mem-ber L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi-omics view on the spatial and temporal changes of astrocytes particularly in the ARC dur-ing different time points of adaptation to a high caloric diet.
... Even though GFAP has been widely used as a standard marker for astrocytes, several studies have reported the existence of astrocytes with undetectable levels of GFAP. Additionally, not all cells in the CNS that express GFAP are astrocytes (Kimelberg 2004;Mishima and Hirase 2010). As an example, layer III and IV astrocytes are devoid of GFAP, although astrocytes appear homogeneously distributed in these regions (Gabbott and Stewart 1987). ...
Astroglial cells actively partner with several cell types to regulate the arrangement of neuronal circuits both in the developing and adult brain. Morphological features of astroglial cells strongly impact their functional interactions, thereby supporting the hypothesis that aberrancies in glial morphology may trigger the onset of neuropsychiatric disorders. Thus, understanding the factors which modulate astroglial shapes and the development of tools to examine them may help to gain valuable insights about the role of astroglia in physiological and pathological brain states.
... Most astrocytes across different brain regions can be marked by Aldehyde dehydrogenase 1 family, member L1 (ALDH1L1) and glutamate transporter (GLAST), while fibrous astrocytes and protoplasmic astrocytes express glial fibrillary acid protein (GFAP) and S100β, respectively. Other proteins, such as Connexins and Aquaporin (AQP4) were regarded as markers of reactive astrocytes under pathological conditions (de Majo et al., 2020;Khakh and Deneen, 2019;Kimelberg, 2004;Savchenko et al., 2000;Verkhratsky and Nedergaard, 2018). Meanwhile, the application of GFAP and S100β is a current routine identifier of astrocytes in the healthy CNS. ...
Astrocytes control many processes of the nervous system in health and disease, and respond to injury quickly. Astrocytes produce neuroprotective factors in the injured brain to clear cellular debris and to orchestrate neurorestorative processes that are beneficial for neurological recovery after traumatic brain injury (TBI). However, astrocytes also become dysregulated and produce cytotoxic mediators that hinder CNS repair by induction of neuronal dysfunction and cell death. Hence, we discuss the potential role of astrocytes in neuropathological processes such as neuroinflammation, neurogenesis, synaptogenesis and blood-brain barrier repair after TBI. Thus, an improved understanding of the dual role of astrocytes may advance our knowledge of post-brain injury recovery, and provide opportunities for the development of novel therapeutic strategies for TBI.
... Apart from the star-shaped cellular body, reflected in the name of this class of neuroglia, characteristic features of the astrocytes include presence of the intermediate filaments (e.g. glial fibrillary acidic protein and vimentin) building their cytoskeleton, perivascular projections and gap junctions, and presence of glutamate and GABA transporters [46][47][48]. Among their characteristic features are also specific properties of energy metabolism and lack of electrical excitability [43,49]. ...
The dynamic development of studies on neuroglia in recent years indicates its previously underestimated role in maintaining proper brain function, both in physiological and pathological conditions. The use of modern research methods such as single-cell techniques as well as in vivo and in vitro models enriched the state of our knowledge. The most important issues regarding the maturation and development of neuroglia include cooperation between glial cell groups and with neurons in neurogenesis, neuroregeneration, (re)myelination and how the early developmental roles of glia contribute to nervous system dysfunction in neurodevelopmental and neurodegenerative disorders. There is still growing evidence emphasizing the importance of astroglia in maintaining the brain physiological homeostasis, regulation of immune response, cerebral blood flow, and involvement in the reactive neurogliosis, precisely adapted to the nature of pathological stimulus and the depth of tissue damage. The important issues related to the function of oligodendrocytes include explanation of the mechanisms of interaction between the glial cells and myelinated axons, important not only in myelination, but also in development of cognitive processes and memory. Further studies are required for understanding the mechanisms of demyelination occurring in several central nervous system (CNS) diseases. An interesting area of research is related with explanation on the NG2 glia function, characterized by significant proliferative potential and ability to differentiate in both in physiological conditions and in pathology, as well as the presence of synaptic neural glial connections, which are especially numerous during development. The increasing knowledge of microglia comprise the presence of specialised subsets of microglia, their role the myelination process and neurovascular unit functioning. We are only beginning to understand how microglia enter the brain and develop distinct functional states during ontogeny. This review summarises the current state of knowledge on the development and role in the CNS of different, heterogeneous cell populations defined by a common term neuroglia.
... Although there are limitations in using these markers, for example GFAP is not expressed in all astrocytes and is not always expressed only in astrocytes [104,105], these are commonly used astrocyte markers with conserved expression across species, and they play differing, yet specific, roles in the reactive astrogliosis response in rats [106][107][108][109]. Taken altogether, these gene expression markers should provide a robust way to identify astrocytes and characterize astrocytic change, if present. Similar results were found in the "look-up" studies, which allow us to explore data sets that use different medication types and at different time-points. ...
Astrocytes have many important functions in the brain, but their roles in CNS disorders and their responses to psychotropic medications are still being elucidated. In this study, we used gene enrichment analysis to assess the relationships between different astrocyte subtypes, neurological and psychiatric diseases, and psychotropic medications. We also carried out qPCR analyses and "look-up" studies to further assess the chronic effects of these drugs on astrocyte marker gene expression. Our bioinformatic analysis identified differential gene enrichment of different astrocyte subtypes in CNS disorders. The "common" astrocyte subtype was the most frequently enriched across disorders, but the highest level of enrichment was found in depression, supporting a role for astrocytes in this disorder. We also identified common enrichment of metabolic and signal transduction-related biological processes in astrocyte subtypes and CNS disorders. However, enrichment of different psychotropic medications, including antipsychotics, antidepressants, and mood stabilizers, was limited in astrocyte subtypes. These results were confirmed by "look-up" studies and qPCR analysis, which also reported little effect of common psychotropic medications on astrocyte marker gene expression, suggesting that astrocytes are not a primary target of these medications. Overall, this study provides a unique view of astrocyte subtypes and the effect of medications on astrocytes in disease, which will contribute to our understanding of their role in CNS disorders and offers insights into targeting astrocytes therapeutically.
... Estas prolongaciones, llamadas gliofilamentos, están ma-yormente compuestas por GFAP, específica para los astrocitos del SNC y que es comúnmente empleada como proteína marcadora. El núcleo de los astrocitos es más claro que el de otras células de la glía y el citoplasma contiene numerosos gránulos de glucógeno (Kimelberg, 2004). La generación y expansión de los astrocitos se completa en gran medida antes del nacimiento, pero la elaboración y maduración de sus finos procesos perisinápticos persiste durante el período activo de sinaptogénesis en el período posnatal (Ullian et al., 2001). ...
Autism spectrum disorder (ASD) is characterized by persistent deficits in communication and social interaction, as well as restrictive or repetitive activities or interests. Its etiology is complex and heterogeneous, and the neurobiological mechanisms that give rise to the clinical phenotype are not yet fully understood. Research points to genetic and environmental factors that affect the developing brain. These advances are consistent with an enhanced understanding of the physiological functions and pathological potential of neuroglia in the central nervous system (CNS) which supports the conclusion of the contribution of these cells in ASD. Therefore, the objective of this article was to briefly review the key risk factors associated with ASD and then explore the contribution of glia in this disorder. The role of astrocytes, microgliocytes and oligodendrocytes in the homeostatic control of the CNS in the immune regulation of the brain and in axonal myelination, as well as malfunction and morphological alterations of these cells in autistic brains are emphasized.
... Astrocytes are specialised cerebral and spinal cord glial cells that ensheath blood vessel with astrocytic end-feet. They provide structural support and as part of the BBB, play a role in tripartite synapse homeostasis and regulate blood flow [285][286][287][288][289][290][291][292]. Astrocytes play a key role in neuron maintenance as well as ionic and osmotic brain homeostasis [293][294][295]. ...
The cerebral vasculature plays a central role in human health and disease and possesses several unique anatomic, functional and molecular characteristics. Despite their importance, the mechanisms that determine cerebrovascular development are less well studied than other vascular territories. This is in part due to limitations of existing models and techniques for visualisation and manipulation of the cerebral vasculature. In this review we summarise the experimental approaches used to study the cerebral vessels and the mechanisms that contribute to their development.
... GFAP is a marker for nearly all of the reactive cells. Nonreactive astroglia contains this protein at a level which is undetectable by immunohistochemical methods [18]. The observed overgrowth of astrocytic processes may be associated with increased activity of CA1 pyramidal neurons. ...
Introduction:
Astrocytes react to microenvironmental changes. Their reactivity is manifested by an increase in glial fibrillary acidic protein (GFAP) and S100β protein levels, hypertrophy and hyperplasia. The aim of the study was to analyse immunoreactive GFAP (GFAP-IR) and S100β (S100β-IR) astrocytes of hippocampal CA1 area in adult rats intragastrically (i.g.) treated with habanero peppers.
Material and methods:
Brains from 10 control rats (group C) and 10 rats receiving oil suspension of habanero fruits for 7 days (group I-7) or 28 days (group II-28) were used. Antibodies against GFAP and S100β were used for immunohistochemistry. Morphology and distribution of astrocytes was evaluated under light microscope and their density was quantitatively analysed.
Results:
In the CA1 hippocampal area of group II-28 rats, GFAP-IR cells with numerous, branched processes were observed. S100β-IR astrocytes had delicate, single processes in comparison with cells without processes observed in groups I-7 and C. In groups I-7 and II-28, GFAP-IR astrocytes' density significantly increased in SR - stratum radiatum of hippocampal CA1 area. In group I-7, a density of cells with the expression of S100β was significantly increased in SO - stratum oriens layer. In group II-28, the density of S100β-IR astrocytes was decreased.
Conclusions:
Habanero peppers administrated to rats, especially for a longer time, caused reactive changes in the astrocytes in hippocampal CA1 area, and thus these glial cells may protect neurons against excitotoxic damage.
... There is general consensus that both GS and Aldh1L1 stain more astrocytes than GFAP, in a region specific manner [28][29][30][31]36]. This is in line with the expression of GS or Aldh1L1 in all regions examined here, in 100% of the GFAP + /Rab6A + cells. ...
Astrocytes contribute to many higher brain functions. A key mechanism in glia-to-neuron signalling is vesicular exocytosis; however, the identity of exocytosis organelles remains a matter of debate. Since vesicles derived from the trans-Golgi network (TGN) are not considered in this context, we studied the astrocyte TGN by immunocytochemistry applying anti-Rab6A. In mouse brain, Rab6A immunostaining is found to be unexpectedly massive, diffuse in all regions, and is detected preferentially and abundantly in the peripheral astrocyte processes, which is hardly evident without glial fibrillary acid protein (GFAP) co-staining. All cells positive for the astrocytic markers glutamine synthetase (GS), GFAP, aldehyde dehydrogenase 1 family member L1 (Aldh1L1), or SRY (sex determining region Y)-box 9 (SOX9) were Rab6A+. Rab6A is excluded from microglia, oligodendrocytes, and NG2 cells using cell type-specific markers. In human cortex, Rab6A labelling is very similar and associated with GFAP+ astrocytes. The mouse data also confirm the specific astrocytic labelling by Aldh1L1 or SOX9; the astrocyte-specific labelling by GS sometimes debated is replicated again. In mouse and human brain, individual astrocytes display high variability in Rab6A+ structures, suggesting dynamic regulation of the glial TGN. In summary, Rab6A expression is an additional, global descriptor of astrocyte identity. Rab6A might constitute an organelle system with a potential role of Rab6A in neuropathological and physiological processes.
... In contrast, colocalization of viral Ag with anti-GFAP Ab was significantly increased in CaMKIIcre:IFNAR fl/fl compared to IFNAR fl/fl mice at day 6 p.i. (Fig. 4D and E). Astrocyte infection may even be underestimated, as anti-GFAP Ab prominently stains astrocyte main stem branches but not cytoplasm or fine processes (12,39,40), thereby excluding viral Ag localized proximally but not overlapping with GFAP. Irrespectively, these results imply that loss . ...
IFN-α/β induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/β functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/β receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/β pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/β and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.
... Historically, the research on IFs in astrocytes did not start with vesicular transport. The first visualization of IFs overlaps to some extent with the discovery of astrocytes, because Golgi's silver staining and its variations (notably Ramón and Cajal's own astrocyte-specific gold sublimate stain) target IFs among other cellular components [15]. Throughout the 20th century, a series of IF-related discoveries were made, mainly in the field of keratins, which coincided with the development of new methodologies and methods for the preparation of biological specimens (for an interesting review, see [16]). ...
Despite the remarkable complexity of the individual neuron and of neuronal circuits, it has been clear for quite a while that, in order to understand the functioning of the brain, the contribution of other cell types in the brain have to be accounted for. Among glial cells, astrocytes have multiple roles in orchestrating neuronal functions. Their communication with neurons by exchanging signaling molecules and removing molecules from extracellular space takes place at several levels and is governed by different cellular processes, supported by multiple cellular structures, including the cytoskeleton. Intermediate filaments in astrocytes are emerging as important integrators of cellular processes. Astrocytes express five types of intermediate filaments: glial fibrillary acidic protein (GFAP); vimentin; nestin; synemin; lamins. Variability, interactions with different cellular structures and the particular roles of individual intermediate filaments in astrocytes have been studied extensively in the case of GFAP and vimentin, but far less attention has been given to nestin, synemin and lamins. Similarly, the interplay between different types of cytoskeleton and the interaction between the cytoskeleton and membranous structures, which is mediated by cytolinker proteins, are understudied in astrocytes. The present review summarizes the basic properties of astrocytic intermediate filaments and of other cytoskeletal macromolecules, such as cytolinker proteins, and describes the current knowledge of their roles in normal physiological and pathological conditions.
... One readout widely used to quantify astrocyte reactivity is the increase of GFAP levels. One concern about this approach is the fact that basal expression of GFAP is variable depending on the brain region, i.e.: hippocampus shows very high expression while cortex almost none (Kimelberg, 2004). There are also technical differences depending where and how GFAP is measured in each study: while some characterized number of GFAP-positive cells (Diniz et al., 2010), others quantified GFAP intensity (Kanaan et al., 2010), area (Rodríguez et al., 2014) or mRNA levels (Nichols et al., 1993). ...
Aging is the strongest risk factor for metabolic, vascular and neurodegenerative diseases. Aging alone is associated with a gradual decline of cognitive and motor functions. Considering an increasing elderly population in the last century, understanding the cellular and molecular mechanisms contributing to brain aging is of vital importance. Recent genetic and transcriptomic findings strongly suggest that glia are the first cells changing with aging. Glial cells constitute around 50% of the total cells in the brain and play key roles regulating brain homeostasis in health and disease. Their essential functions include providing nutritional support to neurons, activation of immune responses, and regulation of synaptic transmission and plasticity. In this review we discuss how glia are altered in the aging brain and whether these alterations are protective or contribute to the age-related pathological cascade. We focus on the major morphological, transcriptional and functional changes affecting glia in a range of systems, including human, non-human primates, and rodents. We also highlight future directions for investigating the roles of glia in brain aging.
... As expected for mature astrocytes, almost all human iPSCderived astrocytes were immunoreactive for GFAP and Vimentin, though not all astrocytes necessarily express GFAP in the human brain tissue (Kettenmann & Verkhratsky, 2011;Kimelberg, 2004). ...
Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1β, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.
... Hétérogénéité astrocytaire : Chez les mammifères, il existe une grande hétérogénéité de phénotype astrocytaire et leur classification fait encore l'objet de débats (Kimelberg, 2004). Cependant deux types principaux d'astrocyte ont tout d'abord été distingués grâce à leurs caractéristiques morphologiques et à leur localisation. ...
La Neuromyélite Optique (NMO) est une maladie auto-immune démyélinisante, rare et grave, du système nerveux central (SNC). Elle est caractérisée par une démyélinisation et une perte axonale ciblant principalement le nerf optique et la moelle épinière. La découverte d'un auto-anticorps (IgG-NMO) dirigé contre l'aquaporine-4 (AQP4), un canal hydrique exprimé par l'astrocyte, a été une étape clé dans la compréhension de la physiopathologie de la NMO, actuellement définie comme une astrocytopathie. La pathogénicité de l'IgG-NMO a été démontrée : il induit une internalisation d'AQP4 et des transporteurs au glutamate, provoquant une altération de la fonction astrocytaire. Cependant les mécanismes permettant de lier la dysfonction astrocytaire aux altérations caractéristiques de la NMO, notamment la démyélinisation, restent méconnus. Les astrocytes sont des cellules gliales essentielles à l'établissement et au maintien de l'homéostasie du SNC. Ils permettent la régulation des flux hydriques et ioniques, le contrôle extracellulaire des neuromédiateurs ainsi que l'apport de métabolites énergétiques aux neurones et aux oligodendrocytes. Ils sont aussi caractérisés par une très forte expression de connexines (Cx), des molécules transmembranaires s'assemblant sous une forme hexamérique : le connexon. Les connexines forment soit des hémicanaux, permettant l'échange de petites molécules entre les milieux intra- et extra-cellulaires, soit des jonctions communicantes par la juxtaposition de connexons appartenant à deux cellules, assurant le couplage intercellulaire avec le passage de petites molécules et d'ions (ATP, glutamate, lactate, calcium). Les fonctions hemicanal et jonction communicante sont fortement régulées en condition physiologique et altérées en condition pathologique, notamment en contexte neuroinflammatoire. Nous émettons l'hypothèse que les IgG-NMO altèrent l'expression et la fonction des connexines, et conduisent ainsi à la production d'un environnement toxique pour les oligodendrocytes et la myéline, et délétère pour le fonctionnement neuronal. Mon projet de thèse avait trois objectifs : i) la caractérisation du phénotype astrocytaire induit par les IgGNMO ; ii) l'identification d'altérations des connexines et leur implication dans la pathologie ; iii) la mise en évidence d'altérations de la transmission synaptique induites par les IgG-NMO et l'implication de connexines dans cet effet. Des modèles de cultures primaires gliales traitées par des IgG-NMO issue d'une cohorte de patients m'ont permis de caractériser le phénotype acquis par les astrocytes, et de proposer le concept d'un astrocyte réactif spécifique de pathologie. Les astrocytes réactifs spécifiques de la NMO induisent un milieu inflammatoire spécifique et toxique, provoquant une démyélinisation. Grâce au développement d'une coculture gliale et neuronale produisant des neurones myélinisés, et à l'utilisation de peptides inhibiteurs des Cx, j'ai pu montrer que les NMO-IgG ont un effet démyélinisant et que celui-ci implique les Cx. La démyélinisation est en effet associée à des modifications structurales et fonctionnelles des Cx astrocytaires, observées à la fois in vitro et dans notre modèle in vivo, le rat-NMO. Enfin, la mise en place d'une étude électrophysiologique en potentiel de champs local sur des tranches d'hippocampe de rats m'a permis d'étudier l'effet des IgG-NMO sur la transmission glutamatergique basale. J'ai pu mettre en évidence un effet dépresseur des IgG-NMO, partiellement bloqué par un inhibiteur de connexines, la carbenoxolone. Comme il a déjà été démontré par des études cliniques dans des pathologies neurodégénératives, l'utilisation de modulateurs de Cx semble être une voie thérapeutique prometteuse afin de prévenir la démyélinisation et les altérations du fonctionnement neuronal de la NMO
... It is noteworthy that although an upregulation of GFAP is usually associated with astrocyte reactivity, this feature itself may not be entirely precise to characterize this process, either in aging or brain disease. This is due to several reasons, such as: the number of GFAP positive cells and the basal GFAP expression level notably vary between different brain regions (Kimelberg, 2004); alteration in GFAP expression is usually region-specific (Rodríguez et al., 2014) and it may depends on the type and stage of the disease, or even the time after injury Diniz et al., 2017;Cunha et al., 2018). ...
Astrocytes, one of the largest glial cell population in the central nervous system (CNS), play a key function in several events of brain development and function, such as synapse formation and function, control of neurotransmitters release and uptake, production of trophic factors and control of neuronal survival. Initially described as a homogenous population, several evidences have pointed that astrocytes are highly heterogeneous, both morphologically and functionally, within the same region, and across different brain regions. Recent findings suggest that the heterogeneity in the expression profile of proteins involved in astrocyte function may predict the selective vulnerability of brain regions to specific diseases, as well as to the age-related cognitive decline. However, the molecular mechanisms underlying these changes, either in aging as well as in brain disease are scarce. Neuroinflammation, a hallmark of several neurodegenerative diseases and aging, is reported to have a dubious impact on glial activation, as these cells release pro- and anti-inflammatory cytokines and chemokines, anti-oxidants, free radicals, and neurotrophic factors. Despite the emerging evidences supporting that reactive astrocytes have a duality in their phenotype, neurotoxic or neuroprotective properties, depending on the age and stimuli, the underlying mechanisms of their activation, cellular interplays and the impact of regional astrocyte heterogeneity are still a matter of discussion. In this review article, we will summarize recent findings on astrocyte heterogeneity and phenotypes, as well as their likely impact for the brain function during aging and neural diseases. We will focus on the molecules and mechanisms triggered by astrocyte to control synapse formation in different brain regions. Finally, we will discuss new evidences on how the modulation of astrocyte phenotype and function could impact the synaptic deficits and glial dysfunction present in aging and pathological states.
... Conversely, GFAP promoterdriven expression of fluorescent reporter proteins can reveal gray matter astrocytes in the cortex, hippocampus, and cerebellum as well as their fine astrocytic processes (Hirrlinger et al., 2005;Nolte et al., 2001). Finally, astrocytes display a wide range of GFAP expression due to their inherent heterogeneity, thus producing variable transgene expression or endogenous protein labeling (Emsley & Macklis, 2006;Grass et al., 2004;Kimelberg, 2004;Matthias et al., 2003;Meng et al., 2015;Nolte et al., 2001) ( Fig. 2A and B). ...
Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contribution to neural processing remains unknown. This is, in part, due to the lack of methods available for astrocyte identification and analysis. Existing strategies employ genetic tools like the astrocyte specific promoters glial fibrillary acidic protein (GFAP) or Aldh1L1 to create transgenic animals with fluorescently labeled astrocytes. Recently, small molecule targeting moieties have enabled the delivery of bright fluorescent dyes to astrocytes. Here, we review methods for targeting astrocytes, with a focus on a recently developed methylpyridinium targeting moiety's development, chemical synthesis, and elaboration to provide new features like light-based spatiotemporal control of cell labeling.
Aim:
Prolonged high-fat diet (HFD) consumption has been shown to impair cognition and depression. The combined effects of HFD and lipopolysaccharide (LPS) administration on those outcomes have never been thoroughly investigated. This study investigated the effects of LPS, HFD consumption, and a combination of both conditions on microglial dysfunction, microglial morphological alterations, synaptic loss, cognitive dysfunction, and depressive-like behaviors.
Methods:
Sixty-four male Wistar rats were fed either a normal diet (ND) or HFD for 12 weeks, followed by single dose-subcutaneous injection of either vehicle or LPS. Then, cognitive function and depressive-like behaviors were assessed. Then, rats were euthanized, and the whole brain, hippocampus, and spleen were collected for further investigation, including western blot analysis, qRT-PCR, immunofluorescence staining, and brain metabolome determination.
Results:
HFD-fed rats developed obese characteristics. Both HFD-fed rats with vehicle and ND-fed rats with LPS increased cholesterol and serum LPS levels, which were exacerbated in HFD-fed rats with LPS. HFD consumption, but not LPS injection, caused oxidative stress, blood-brain barrier disruption, and decreased neurogenesis. Both HFD and LPS administration triggered an increase in inflammatory genes on microglia and astrocytes, increased c1q colocalization with microglia, and increased dendritic spine loss, which were exacerbated in the combined conditions. Both HFD and LPS altered neurotransmitters and disrupted brain metabolism. Interestingly, HFD consumption, but not LPS, induced cognitive decline, whereas both conditions individually induced depressive-like behaviors, which were exacerbated in the combined conditions.
Conclusions:
Our findings suggest that LPS aggravates metabolic disturbances, neuroinflammation, microglial synaptic engulfment, and depressive-like behaviors in obese rats.
Neurons are highly specialized for circuit activity. Their unique structure and function make them very vulnerable to small fluctuations in the composition of the extracellular space. A very important process is synaptic integration, which is the most energy-consuming process of the neuronal circuit. As the synapses are very often far from the cell body, a dynamic energy supply is a problem. For these and other challenges, the brain has an elaborate system of support or glial cells which in addition can act as partners in signaling pathways. The major group of glia are astrocytes, which have their own elaborate structure with domains and constitute a syncytium. They also cover the blood–brain barrier with endfeet. Their architecture destines them to be the prime partner of neurons in homeostasis and signaling. Another glial cell group is oligodendrocytes and their precursor cells. Oligodendrocytes specialize for myelination, whereas oligodendrocyte precursor cells are more complicated as they are on the receiving end of neuronal presynaptic endings. Finally, microglia are macrophages trapped in the brain after the establishment of the blood–brain barrier. They adapted to this peculiar environment by not only playing the part of a resident macrophage waiting for pathological challenges but also being involved in neuronal signaling. Three fluid systems are irrigating the brain. The blood supply thins out into fine processes, whose endothelial cells are the location of specific exchange systems and of the blood–brain barrier. The cerebrospinal fluid is created by the choroid plexus and migrates by bulk flow. It is in free exchange with the parenchyma and empties into the lymph and venous system, depending on pressure gradients. The glymphatic system is a convection system, from the perivascular arterial space through the glial syncytium and extracellular space toward several exit passages, venous system, and lymphatic system and on the way possibly mixing with cerebrospinal fluid. It is dependent on aquaporin channels and the dimensions of the extracellular space. That space is almost doubled during sleep, which eases the removal of waste products like β-amyloid peptide.
Astrocytes are key cells in the central nervous system. They are involved in many important functions under physiological and pathological conditions. As part of neuroglia, they have been recognised as cellular elements in their own right. The name astrocyte was first proposed by Mihaly von Lenhossek in 1895 because of the finely branched processes and star-like appearance of these particular cells. As early as the late 19th and early 20th centuries, Ramon y Cajal and Camillo Golgi had noted that although astrocytes have stellate features, their morphology is extremely diverse. Modern research has confirmed the morphological diversity of astrocytes both in vitro and in vivo and their complex, specific, and important roles in the central nervous system. In this review, the functions of astrocytes and their roles are described.
Hypothalamic astrocytes are particularly affected by energy‐dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nucleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following exposure to a high‐fat high‐sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile dependent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single‐cell sequencing, we observed that a HFHS diet dictates time‐ and cell‐ specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pattern compared to other cell types. Lastly, we topographically and molecularly characterized astrocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family member L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi‐omics view on the spatial and temporal changes of astrocytes particularly in the ARC during different time points of adaptation to a high calorie diet. MAIN POINTS: Hypothalamic astrocytes are predominantly altered in diet‐induced obese mice. Hypercaloric diet rapidly rearranges the molecular state of ARC astrocytes, inducing the expression of GFAP and Aldh1L1 in a time‐ and region‐dependent manner.
Glia are widely distributed in the central nervous system (CNS) and are closely related to cell metabolism, signal transduction, support, cell migration and other nervous system development processes and functions. Glial development is complex and essential, including the processes of proliferation, differentiation and migration, and requires precise regulatory networks. Noncoding RNAs can be deeply involved in glial development through gene regulation. Here, we review the regulatory roles of noncoding RNAs in glial development. We briefly describe the classification and functions of noncoding RNAs and focus on miRNAs and lncRNAs, which have been reported to participate extensively during glial formation. The highlight of this summary is that miRNAs and lncRNAs can participate in and regulate the signaling pathways of glial development. The review not only describes how noncoding RNAs participate in nervous system development but also explains the processes of glial development, providing a foundation for subsequent studies on glial development and new insights into the pathogeneses of related neurological diseases.
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Manganese in the diet is nutritionally essential for normal physiologic functioning. However, excessive exposure to manganese has been associated with developmental, neurodegenerative and other disorders.
The book comprehensively covers the toxicology of manganese. Leading investigators provide perspectives from toxicology, neuroscience, nutrition, molecular biology and risk assessment disciplines and chapters cover the toxicokinetics, toxicodynamic interactions and health effects of manganese, as well as its potential role in neurodegenerative diseases. A large section devoted to health effects presents the latest research that associates manganese exposure to potential human diseases.
Any scientists, health professional or regulator involved with metal exposure and toxicology should find this volume essential reading. Students and researchers in neurotoxicology will also find this book a useful reference.
KAUL, D., S.G. Schwab, N. Mechawar and N. Matosin. How Stress Physically Re-shapes the Brain: Impact on Brain Cell Shapes, Numbers and Connections in Psychiatric Disorders…NEUROSCI BIOBEHAV REV XX(X) XXX-XXX, 2020.-Severe stress is among the most robust risk factors for the development of psychiatric disorders. Imaging studies indicate that life stress is integral to shaping the human brain, especially regions involved in processing the stress response. Although this is likely underpinned by changes to the cytoarchitecture of cellular networks in the brain, we are yet to clearly understand how these define a role for stress in human psychopathology. In this review, we consolidate evidence of macro-structural morphometric changes and the cellular mechanisms that likely underlie them. Focusing on stress-sensitive regions of the brain, we illustrate how stress throughout life may lead to persistent remodelling of the both neurons and glia in cellular networks and how these may lead to psychopathology. We support that greater translation of cellular alterations to human cohorts will support parsing the psychological sequalae of severe stress and improve our understanding of how stress shapes the human brain. This will remain a critical step for improving treatment interventions and prevention outcomes.
S100B is an EF-hand type Ca2+-binding protein of the S100 family, known to support neurogenesis and to promote the interactions between brain’s nervous and immune systems. Here, we characterized the expression of S100B in the mouse olfactory bulb, a neurogenic niche comprising mature and adult-born neurons, astrocytes, oligodendrocytes and microglia. Besides astrocytes, for which S100B is a classical marker, S100B was also expressed in NG2 cells and, surprisingly, in APC-positive myelinating oligodendrocytes but not in mature/adult-born neurons or microglia. Various layers of the bulb differed substantially in the composition of S100B-positive cells, with the highest fraction of the APC-positive oligodendrocytes found in the granule cell layer. Across all layers, ∼50 % of NG2 cells were S100B-negative. Finally, our data revealed a strong correlation between the fraction of myelinating oligodendrocytes among the S100B-positive cells and the oligodendrocyte density in different brain areas, underscoring the importance of S100B for the establishment and maintenance of myelin sheaths.
Astrocytes are the primary homeostatic cells of the central nervous system, essential for normal neuronal development and function, metabolism and response to injury and inflammation. Here, we review postmortem studies examining changes in astrocytes in subjects diagnosed with the neuropsychiatric disorders schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BPD). We discuss the astrocyte-related changes described in the brain in these disorders and the potential effects of psychotropic medication on these findings. Finally, we describe emerging tools that can be used to study the role of astrocytes in neuropsychiatric illness.
Macroglia, comprising astrocytes and oligodendroglial lineage cells, have long been regarded as uniform cell types of the central nervous system (CNS). Although regional morphological differences between these cell types were initially described after their identification a century ago, these differences were largely ignored. Recently, accumulating evidence suggests that macroglial cells form distinct populations throughout the CNS, based on both functional and morphological features. Moreover, with the use of refined techniques including single-cell and single-nucleus RNA sequencing, additional evidence is emerging for regional macroglial heterogeneity at the transcriptional level. In parallel, several studies revealed the existence of regional differences in remyelination capacity between CNS grey and white matter areas, both in experimental models for successful remyelination as well as in the chronic demyelinating disease multiple sclerosis (MS). In this review, we provide an overview of the diversity in oligodendroglial lineage cells and astrocytes from the grey and white matter, as well as their interplay in health and upon demyelination and successful remyelination. In addition, we discuss the implications of regional macroglial diversity for remyelination in light of its failure in MS. Since the etiology of MS remains unknown and only disease-modifying treatments altering the immune response are available for MS, the elucidation of macroglial diversity in grey and white matter and its putative contribution to the observed difference in remyelination efficiency between these regions may open therapeutic avenues aimed at enhancing endogenous remyelination in either area.
Neuron-supporting glial cells display a considerable variability in phenotype and function. The first part of the chapter describes general principles of glial cell phenotype and distribution including evolution of glia-mediated neuronal support, glial morphology according to the types of cell processes, glial morphology in development, and glioneuronal domains of information processing at various hierarchical levels. The second part describes in more detail the various phenotypes and functions of glial cells in the mammalian central and peripheral nervous systems.
Background
Astrocytes are the main cellular constituent in the central nervous system. Astrocyte cultures from rodent brains are most commonly used in the experimental practice. However, important differences between rodent and human astrocytes exist. The aim of this study was to develop an improved protocol for routine preparation of primary astrocyte culture from adult human brain, obtained after trauma.
New Method
Tissue obtained during neurotrauma operation was mechanically decomposed and centrifuged. The cell sediment was resuspended in cell culture medium, plated in T25 tissue flasks and incubated for one month at 37 °C in 5 % CO2. The medium was replaced twice weekly and microglia were removed. Once confluent, the purity of cultures was assessed. The culture was characterised immunocytochemically for specific astrocytic markers (GFAP, GLAST and S100B). Cell morphology was examined through the actin cytoskeleton labelling with fluorescent phalloidin.
Results
Under basal conditions, adult astrocytes exhibited astrocyte-specific morphology and expressed specific markers. Approximately 95 % of cells were positive for the main glial markers (GFAP, GLAST, S100B).
Comparison with Existing Method
We established an easy and cost-effective method for a highly enriched primary astrocyte culture from adult human brain.
Conclusion
The isolation technique provides sufficient quantities of isolated cells. The culture obtained in this study exhibits the biochemical and physiological properties of astrocytes. It may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, novel therapeutic targets, cell therapy experiments, as well as investigating compounds involved in cytotoxicity and cytoprotection.
La synapse est le lieu de communication entre les neurones à l'origine de nos capacités cognitives. Les mutations des gènes codant pour des protéines synaptiques sont responsables des maladies neurodéveloppementales appelées synaptopathies, recouvrant un large spectre de pathologies, de la déficience intellectuelle aux troubles du spectre autistique. Cependant, il est actuellement établi que les neurones ne sont pas les seuls acteurs au niveau de la synapse. Les astrocytes jouent également un rôle essentiel dans la mise en place du réseau neuronal et le fonctionnement de la synapse. Ils assurent aussi l'homéostasie ionique synaptique et sont capables de sécréter des glio-transmetteurs qui modulent l'activité synaptique. Oligophrénine-1 (OPHN1) est un gène associé à la déficience intellectuelle liée à l'X chez l'Homme. OPHN1 est une protéine synaptique dont les fonctions neuronales sont bien connues. La protéine peut directement interagir avec le cytosquelette d'actine et joue un rôle dans la formation et la maturation des épines dendritiques. Cette protéine est aussi exprimée dans les astrocytes mais sa fonction astrocytaire n'est pas connue. A l'aide d'un modèle KO de souris pour Ophn1, nous avons mis en évidence les conséquences de l'absence d'Ophn1 dans les astrocytes. Nous avons démontré que la délétion d'OPHN1 altère la migration et la morphologie des astrocytes in vitro. Sachant qu'Ophn1 est capable d'inactiver la GTPase RhoA, nous avons utilisé un inhibiteur de la voie RhoA/ROCK pour retrouver un phénotype de migration normal. In vivo nous avons choisi un modèle de cicatrisation gliale cortical afin de pouvoir observer la migration et la morphologie des astrocytes au niveau de la cicatrice. Nous avons observé que la délétion d'Ophn1 altérait la cicatrisation gliale et que les astrocytes à proximité de la cicatrice était moins ramifiés. L'ensemble de ces résultats nous permet de constater que les astrocytes sont altérés dans notre modèle murin de déficience intellectuelle liée à l'X. De plus, le KO conditionel astrocytaire mis en place nous permettra à l'avenir d'étudier les conséquences de la perte d'OPHN1 uniquement dans les astrocytes, et de comprendre la contribution astrocytaire dans la physiopathologie de cette maladie neuro-développementale.
Astrocytes are now known to play crucial roles in the central nervous system, supporting and closely interacting with neurons and therefore able to modulate brain function. Both human postmortem studies in brain samples from patients diagnosed with Major Depressive Disorder and from animal models of depression reported numerical and morphological astrocytic changes specifically in the hippocampus. In particular, these studies revealed significant reductions in glial cell density denoted by a decreased number of S100B-positive cells and a decrease in GFAP expression in several brain regions including the hippocampus. To reveal plastic astrocytic changes in the context of recurrent depression, we longitudinally assessed dynamic astrocytic alterations (gene expression, cell densities and morphologic variations) in the hippocampal dentate gyrus under repeated exposure to unpredictable chronic mild stress (uCMS) and upon treatment with two antidepressants, fluoxetine and imipramine. Both antidepressants decreased astrocytic complexity immediately after stress exposure. Moreover, we show that astrocytic alterations, particularly an increased number of S100B-positive cells, are observed after recurrent stress exposure. Interestingly, these alterations were prevented at the long-term by either fluoxetine or imipramine treatment.
Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer’s disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrP Sc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states.
Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominately on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only ∼15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.
We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology. GLIA 33:72–86, 2001. © 2000 Wiley-Liss, Inc.
Immunofluorescence and immunoperoxidase techniques were used to localize a cell surface chondroitin-sulfate proteoglycan antigen, termed NG2, in the developing and adult rat cerebellum. In the adult, both polyclonal and monoclonal anti-NG2 antibodies labeled cells throughout the cerebellar cortex, with the labeled cells being especially prominent in the molecular layer. The labeled cells had small, irregularly shaped cell bodies from which thin highly branched processes radiated in a stellate array. The NG2-labeled cells were not labeled with antibodies against glial fibrillary acidic protein (GFAP), vimentin, or S-100 protein, intracellular markers for astrocytes. However, electron microscopic immunocytochemical analysis of NG2 immunoreactive cells revealed a cell morphology consistent with that of protoplasmic astrocytes. Labeled cell bodies contained a thin rim of organelle-poor cytoplasm surrounding a euchromatic nucleus. Thick processes originating from the cell soma tapered to form thin branches with highly irregular surface contours that extended between adjacent neuronal elements. The labeled processes did not form synapses in the neuropil, and no synaptic profiles onto anti-NG2-labeled cell bodies or processes were observed. Thus, we conclude that the NG2 antigen is a cell surface marker for a class of smooth protoplasmic astrocytes. Immunoreactive cells were seen in the developing cerebellum beginning at embryonic day 16. The number of labeled cells increased during the early stages of cerebellar development, reaching a peak at about postnatal day (PND) 4 or 5 and declining thereafter. In the developing cerebellum, labeled cells lying within the forming molecular layer resembled the cells seen in the adult, whereas cells lying deeper within the folia had an immature appearance with fewer processes and less branching. This apparent gradient of morphological maturation suggests that an interaction with parallel fibers in the developing molecular layer may play a role in the terminal cytodifferentiation of the NG2-labeled smooth protoplasmic astrocytes.
Recent studies show that glutamate transporter-mediated currents occur in astrocytes when glutamate is released from hippocampal synapses. These transporters remove excess glutamate from the extracellular space, thereby facilitating synaptic input specificity and preventing neurotoxicity. Little is known about the position of astrocytic processes at hippocampal synapses. Serial electron microscopy and three-dimensional analyses were used to investigate structural relationships between astrocytes and synapses in stratum radiatum of hippocampal area CA1 in the mature rat in vivo and in slices. Only 57 +/- 11% of the synapses had astrocytic processes apposed to them. Of these, the astrocytic processes surrounded less than half (0.43 +/- 22) of the synaptic interface. Other studies suggest that astrocytes extend processes toward higher concentrations of glutamate; thus the presence of astrocytic processes at particular hippocampal synapses might signal which ones are releasing glutamate. The distance between nearest neighboring synapses was usually (approximately 95%) <1 microgram. Astrocytic processes occurred along the extracellular path between 33% of the neighboring synapses, neuronal processes occurred along the path between another 66% of the neighboring synapses, and only 1% of the synapses were close enough such that neither astrocytic nor neuronal processes occurred between them. These morphological arrangements suggest that the glutamate released at approximately two-thirds of hippocampal synapses might diffuse to other synapses, unless neuronal glutamate transporters are more effective than previously reported. The findings also suggest that physiological recordings made from hippocampal astrocytes do not uniformly sample the glutamate released from all hippocampal synapses.
Fast excitatory neurotransmission in the central nervous system occurs at specialized synaptic junctions between neurons, where a high concentration of glutamate directly activates receptor channels. Low-affinity AMPA (alpha-amino-3-hydroxy-5-methyl isoxazole propionic acid) and kainate glutamate receptors are also expressed by some glial cells, including oligodendrocyte precursor cells (OPCs). However, the conditions that result in activation of glutamate receptors on these non-neuronal cells are not known. Here we report that stimulation of excitatory axons in the hippocampus elicits inward currents in OPCs that are mediated by AMPA receptors. The quantal nature of these responses and their rapid kinetics indicate that they are produced by the exocytosis of vesicles filled with glutamate directly opposite these receptors. Some of these AMPA receptors are permeable to calcium ions, providing a link between axonal activity and internal calcium levels in OPCs. Electron microscopic analysis revealed that vesicle-filled axon terminals make synaptic junctions with the processes of OPCs in both the young and adult hippocampus. These results demonstrate the existence of a rapid signalling pathway from pyramidal neurons to OPCs in the mammalian hippocampus that is mediated by excitatory, glutamatergic synapses.
The developing central nervous system of vertebrates contains an abundant cell type designated radial glial cells. These cells are known as guiding cables for migrating neurons, while their role as precursor cells is less clear. Since radial glial cells express a variety of astroglial characteristics and differentiate as astrocytes after completing their guidance function, they have been considered as part of the glial lineage. Using fluorescence-activated cell sorting, we show here that radial glial cells also are neuronal precursors and only later, after neurogenesis, do they shift towards an exclusive generation of astrocytes. These results thus demonstrate a novel function for radial glial cells, namely their ability to generate two major cell types found in the nervous system, neurons and astrocytes.
Whether astrocytes predominantly express ohmic K(+) channels in vivo, and how expression of different K(+) channels affects [K(+)](o) homeostasis in the CNS have been long-standing questions for how astrocytes function. In the present study, we have addressed some of these questions in glial fibrillary acidic protein [GFAP(+)], freshly isolated astrocytes (FIAs) from CA1 and CA3 regions of P7-15 rat hippocampus. As isolated, these astrocytes were uncoupled allowing a higher resolution of electrophysiological study. FIAs showed two distinct ion current profiles, with neither showing a purely linear I-V relationship. One population of astrocytes had a combined expression of outward potassium currents (I(Ka), I(Kd)) and inward sodium currents (I(Na)). We term these outwardly rectifying astrocytes (ORA). Another population of astrocytes is characterized by a relatively symmetric potassium current pattern, comprising outward I(Kdr), I(Ka), and abundant inward potassium currents (I(Kin)), and a larger membrane capacitance (C(m)) and more negative resting membrane potential (RMP) than ORAs. We term these variably rectifying astrocytes (VRA). The I(Kin) in 70% of the VRAs was essentially insensitive to Cs(+), while I(Kin) in the remaining 30% of VRAs was sensitive. The I(Ka) of VRAs was most sensitive to 4-aminopyridine (4-AP), while I(Kdr) of ORAs was more sensitive to tetraethylammonium (TEA). ORAs and VRAs occurred approximately equally in FIAs isolated from the CA1 region (52% ORAs versus 48% VRAs), but ORAs were enriched in FIAs isolated from the CA3 region (71% ORAs versus 29% VRAs), suggesting an anatomical segregation of these two types of astrocytes within the hippocampus. VRAs, but not ORAs, showed robust inward currents in response to an increase in extracellular K(+) from 5 to 10 mM. As VRAs showed a similar current pattern and other passive membrane properties (e.g., RMP, R(in)) to "passive astrocytes"in situ (i.e., these showing linear I-V curves), such passive astrocytes possibly represent VRAs influenced by extensive gap-junction coupling in situ. Thus, our data suggest that, at least in CA1 and CA3 regions from P7-15 rats, there are two classes of GFAP(+) astrocytes which possess different K(+) currents. Only VRAs seem suited to uptake of extracellular K(+) via I(Kin) channels at physiological membrane potentials and increases of [K(+)](o). ORAs show abundant outward potassium currents with more depolarized RMP. Thus VRAs and ORAs may cooperate in vivo for uptake and release of K(+), respectively.
We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology.
Neurogenesis in the dentate gyrus of the hippocampus persists throughout life in many vertebrates, including humans. The progenitors of these new neurons reside in the subgranular layer (SGL) of the dentate gyrus. Although stem cells that can self-renew and generate new neurons and glia have been cultured from the adult mammalian hippocampus, the in vivo primary precursors for the formation of new neurons have not been identified. Here we show that SGL cells, which express glial fibrillary acidic protein and have the characteristics of astrocytes, divide and generate new neurons under normal conditions or after the chemical removal of actively dividing cells. We also describe a population of small electron-dense SGL cells, which we call type D cells and are derived from the astrocytes and probably function as a transient precursor in the formation of new neurons. These results reveal the origins of new neurons in the adult hippocampus.
We have shown previously that process-bearing GFAP+ astrocytes freshly isolated from rat hippocampus CA1 and CA3 regions are heterogeneous in ion channel expression and K(+) uptake capabilities, such that two distinct populations of astrocytes can be described (Zhou and Kimelberg, 2000). In the present study, we report that glutamate transporter (GT) currents can only be measured from one type of these freshly isolated hippocampal CA1 astrocytes [variably rectifying astrocytes (VRAs)] but were not detectable in the second type of astrocyte [outwardly rectifying astrocytes (ORAs)]. The GT currents showed a strict Na(+) dependency and high affinity for glutamate (EC(50) of 4 +/- 1.1 microm). The astrocytes lacking GT currents (ORAs) showed an AMPA receptor current density (55 pA/pF) that was 42-fold higher than VRAs (1.3 pA/pF). In contrast, the GABA(A) currents were of comparable current density in both types. The specificity of these differences makes it unlikely that they are attributable to preparative damage. Therefore, these findings strongly indicate that, within a single region of the hippocampus, GFAP+ astrocytes comprise a functionally diverse population that are qualitatively different in their functional glutamate transporter and quantitatively different in their functional AMPA receptor expression. This heterogeneity implies that GFAP+ astrocytes may participate in or modulate glutamate synaptic transmission differently.
Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominantly on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only approximately 15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.
Radial glial cells, ubiquitous throughout the developing CNS, guide radially migrating neurons and are the precursors of astrocytes. Recent evidence indicates that radial glial cells also generate neurons in the developing cerebral cortex. Here we investigated the role of the transcription factor Pax6 expressed in cortical radial glia. We showed that radial glial cells isolated from the cortex of Pax6 mutant mice have a reduced neurogenic potential, whereas the neurogenic potential of non-radial glial precursors is not affected. Consistent with defects in only one neurogenic lineage, the number of neurons in the Pax6 mutant cortex in vivo is reduced by half. Conversely, retrovirally mediated Pax6 expression instructs neurogenesis even in astrocytes from postnatal cortex in vitro. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells.
Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced “regional adaptation” of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently. J. Comp. Neurol. 431:460–480, 2001. © 2001 Wiley-Liss, Inc.
Elsewhere, I have presented the argument that the action that is carried out at the level of the neuronal articulation between the nervous termination and the dendrites and cellular bodies of successive neurons is of a chemical nature. Every nervous termination suffers a chemical modification and this chemical modification in turn gives stimulus to another neurone. If this is true, the interneuronal articulation would be the center of the chemical exchange, and this would comprise therefore in all the most proximal, vacant interstitial spaces, a region for infiltration of the protoplasmic prolongations or feathery extensions of the neuroglia, perhaps with the purpose of collecting and instantly processing the smallest amount of waste product. (E. Lugaro, 1907)
The cytology of the postmitotic migratory granule cell and its relationship to Bergmann glial processes was examined with Golgi staining and electron microscopy in the three cardinal planes in the developing cerebellar cortex of Macacus rhesus at various late fetal and early postnatal ages. After final mitosis the granule cell body transforms from a nearly round shape in the superficial zone of the external granular layer to a horizontal bipolar form with elongated processes oriented longitudinally to the folium, at the outer border of the molecular layer. Another descending process develops, and the cell soma becomes a pyramid flattened in the plane longitudinal to the folium. The nucleus moves into the descending process and the cell soma assumes a vertically oriented spindle shape while migrating among previously formed parallel fibers deeper in the molecular layer, and finally attains a round shape again when it lies deep to the Purkinje cell layer. During these transformations, the cell cytoplasm becomes more voluminous and contains a prominent Golgi apparatus, numerous free ribosomes, mitochondria, multivesicular and dense bodies, and fascicles of microtubules Longitudinally oriented microtubules concentrated in the vertical leading process disappear by the time the cell soma enters the granular layer. The slender trailing process loss most of its cytoplasmic organelles, acquires microtubules and together with the horizontal processes forms the characteristic T-shaped axon. The axon forms synapses with Purkinje and stellate cell dendrites at a time when other granule cells are still migrating among them.
Radial glial cells play a major guidance role for migrating neurons during central nervous system (CNS) histogenesis but also play many other crucial roles in early brain development. Being among the earliest cells to differentiate in the early CNS, they provide support for neuronal migration during embryonic brain development; provide instructive and neurotrophic signals required for the survival, proliferation, and differentiation of neurons; and may be multipotential progenitor cells that give rise to various cell types, including neurons. Radial glial cells constitute a major cell type of the developing brain in numerous nonmammalian and mammalian vertebrates, increasing in complexity in parallel with the organization of the nervous tissue they help to build. In mammalian species, these cells transdifferentiate into astrocytes when neuronal migration is completed, whereas, in nonmammalian species, they persist into adulthood as a radial component of astroglia. Thus, our perception of radial glia may have to change from that of path-defining cells to that of specialized precursor cells transiently fulfilling a guidance role during brain histogenesis. In that respect, their apparent change of phenotype from radial fiber to astrocyte probably constitutes one of the most common transdifferentiation events in mammalian development. J. Neurosci. Res. 61:357–363, 2000. © 2000 Wiley-Liss, Inc.
Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced “regional adaptation” of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently. J. Comp. Neurol. 431:460–480, 2001. © 2001 Wiley-Liss, Inc.
Radial glial cells (epithelial cells of Ramón y Cajal) impregnated by a modified del Rio Hortega rapid Golgi method were studied in the occipital lobes of 38 rhesus monkeys from embryonic day 48 (E48) to birth which occurs at E165 and in 27 postnatal animals to day 365 (P365). Some radial glial cells are already recognized at E48 by their bipolar shape and elongated radial fiber, which terminates with characteristic endfeet on the walls of blood vessels or at the pial surface. At slightly older ages-between E60 and E70-all cells spanning the cerebral wall develop lamellate expansions along their radial fiber and their endfeet become PAS positive. After E60, some radial glia detach from the ventricular surface and their somas become displaced outwards in the cerebral wall. After this age, radial glial cells are easily distinguished from migrating neurons by their larger oval nucleus located in the ventricular or subventricular zone, radial fiber extending outwards to the pial surface where it terminates in one or more endfeet, and the delicate lamellate expansions on both radial fiber and soma.
Displaced radial glial cells have more closely packed lamellate expansions and display a range of transitional shapes leading to either fibrous or protoplasmic astrocytes. Between E95 and E140, when neuron migration to the visual cortex tapers off, perikarya of displaced radial glial cells form a conspicuous band at the outer border of the subventricular zone. Numerous transitional forms are present in the cortical plate at this age. After birth, fewer radial glial fibers are present in occipital lobe and their length is difficult to determine in the convoluted lateral cerebral wall expanded up to 10–20 mm. However, at P7 and P20, many radial fibers still span the medial cerebral wall in the depth of the calcarine fissure where it remains less than 2 mm thick. Even here, no fibers spanning the cerebral wall were seen in 17 animals from P50 to P200 despite the presence of well-impregnated transitional forms situated near the lateral ventricle and myriad astrocytes dispersed throughout the hemisphere. By P365, end of the first year, the few short remaining radial fibers belong to ependymal cells or mature astrocytes while all immature transitional forms have disappeared.
Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.
Astrocytes have been classically described based on morphological characteristics as either protoplasmic or fibrous. However, the appearance of astrocytes at the light microscopic level following immunoreaction with glial filament acidic protein antisera or Cajal's gold-chloride method is markedly different from the morphology of Golgi-impregnated or horseradish peroxidase-filled astrocytes. The present study combines immunohistochemistry using glial fibrillary acidic protein antisera and a cellular suspension of structurally intact astrocytes isolated from the rat cerebral cortex to demonstrate that the distribution of glial filaments is limited to major astrocytic processes. Because the sheet-like processes which decorate the major branches of protoplasmic astrocytes do not contain glial filaments, the entire protoplasmic astrocyte is not visible when viewed in situ in the gray matter of the cerebral cortex. Thus staining techniques that have a propensity for the glial filaments such as antisera to glial fibrillary acidic protein and the gold-chloride method provide only a skeletal outline of the major processes of the protoplasmic astrocyte. On the other hand, fibrous astrocytes which do not have feathery decorations on major processes are visible in the cerebral cortex in their structural entirety. The present investigation also offers additional evidence of the specificity of glial fibrillary acidic protein antisera for astrocytes.
This paper deals with changes in the arrangement of microfilaments at various stages during the transformation of astroblasts into reactive astrocytes in the presence of dibutyryl 3',5'-cyclic adenosine monophosphate in vitro. When cultures of two-week-old mouse astroblasts are treated with dibutyryl cyclic AMP, drastic changes occur in cell shape and in the organization of microfilaments, resulting in cells that closely resemble reactive astrocytes in vivo. A thick, prominent ring of microfilaments in such cells which stains strongly with 7-nitrobenz-2 oxa-1,3-diazole-phallacidin, delineates the perikaryon. Electron microscope examination showed that the ring is composed of many smaller bundles of microfilaments running parallel to each other. Prominent bundles of microfilaments radiate from the cell body into the cell processes. Based on the observation of intermediate forms, we propose that the microfilament ring may be important in the development of cell processes in reactive astrocytes.
The evolution of concepts concerning the identity and the functions of neuroglia is traced. Some of the main ideas in the works of Virchow, Deiters, Golgi, Lenhossék, Lugaro, Ramón y Cajal, del Río-Hortega, Achúcarro, Penfield, and others are highlighted.
How the immense population of neurons that constitute the human cerebral neocortex is generated from progenitors lining the
cerebral ventricle and then distributed to appropriate layers of distinctive cytoarchitectonic areas can be explained by the
radial unit hypothesis. According to this hypothesis, the ependymal layer of the embryonic cerebral ventricle consists of
proliferative units that provide a proto-map of prospective cytoarchitectonic areas. The output of the proliferative units
is translated via glial guides to the expanding cortex in the form of ontogenetic columns, whose final number for each area
can be modified through interaction with afferent input. Data obtained through various advanced neurobiological techniques,
including electron microscopy, immunocytochemistry, [3H]thymidine and receptor autoradiography, retrovirus gene transfer,
neural transplants, and surgical or genetic manipulation of cortical development, furnish new details about the kinetics of
cell proliferation, their lineage relationships, and phenotypic expression that favor this hypothesis. The radial unit model
provides a framework for understanding cerebral evolution, epigenetic regulation of the parcellation of cytoarchitectonic
areas, and insight into the pathogenesis of certain cortical disorders in humans.
Rabbit retinal glia was studied by light microscopy of both stained sections of frozen retinae and enzymatically isolated cells. In the vast majority of this tissue, except for a small region around the optic nerve head, the glia consists solely of radial glia, i.e. Müller cells whose morphology was found to depend markedly on their topographic localization within the retina. Müller cells in the periphery are short and have thick vitreal processes bearing a single large endfoot. Central Müller cells are long and slender; through the thickening nerve fibre layer they send vitreal processes which are subdivided into several fine branches ending with multiple small endfeet. Müller cells in the retinal centre are far more closely packed than those in the periphery; everywhere, however, a constant ratio of Müller cells: neurons of about 1:15 was found, except for the juxta-optic nerve head region where this ratio is slightly reduced. Where the central retina reaches a thickness requiring Müller cell lengths of more than 130 micron, additional non-radial glial cells occur within the nerve fibre layer. The majority of these cells seem to be astrocytes. Their number per retinal area increases with the thickening of both the whole retina and the nerve fibre layer. The occurrence of these non-radial glial cells leads to an enhancement of the glia:neuron index in the retinal centre. Possible mechanisms of physiological control of gliogenesis are discussed.
The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.
Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-day-old rats frequently showed increased intracellular Ca2+ concentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2+ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2+ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.
Green fluorescent protein (hGFP-S65T) was expressed in transgenic mice under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Tissues from two independent transgenic lines were characterized by Northern blot analysis and by confocal microscopy. The expression pattern in these two lines was identical in all tissues examined, and similar to that found previously with a lacZ transgene driven by the same promoter. Bright fluorescence was observed in the cell bodies and processes of unfixed or fixed astrocytes, using both whole mount and brain slice preparations, from multiple areas of the central nervous system. However, in contrast to GFAP-lacZ transgenics, retinal Müller cells expressed the GFP transgene in response to degeneration of neighboring photoreceptors. These data indicate that the 2.2-kb hGFAP promoter contains sufficient regulatory elements to direct expression in Müller cells, and that GFP is a suitable reporter gene for use in living preparations of the mammalian nervous system. Such mice should prove useful for studies of dynamic changes in astrocyte morphology during development, and in response to physiological and pathological conditions.
For several decades, the reactive gliosis that occurs after an injury to the CNS has been considered one of the major impediments to axonal regeneration. Nevertheless, recent studies have suggested that in certain conditions, reactive astrocytes may provide a permissive substratum to support axonal regrowth. The important criteria, allowing for the distinction between permissive and non-permissive gliosis, are the ultrastructural 3D organization of the scar and more importantly the recognition molecules expressed by reactive astrocytes. Reactive astrocytes express surface molecules and produce various neurotrophic factors and cytokines. The latter in turn might modulate the production of recognition molecules by reactive astrocytes, allowing them to support post-lesional axonal regrowth. Although numerous recent articles have focused on cytokines and cell adhesion molecules, scant attention has been paid to reactive astrocytes. Reactive astrocytes should be considered a key element, like neurons, of a dynamic environment, thus forming with neurons a functional unit involved in homeostasis, plasticity and neurotransmission. Attempts are in progress to identify molecular markers for reactive astrocytes.
In order to establish the relative distribution of a GFAP-negative population of astrocytes, and its change in gliotic tissue, sections of the stratum radiatum of the CA1 hippocampal layer of male, adult, Wistar rats were analyzed by immunocytochemical methods. Ten micrometer-thick sections were triple-stained to detect nuclei, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). In another set of experiments, the rats received a one-time intraperitoneal injection of kainic acid that caused epileptic seizures. With the use of a behavioral protocol, animals with substantial neuronal loss in the pyramidal layer were selected. Five days after the injection these rats were analyzed similarly to control rats. We find that GFAP-positive cells are a subpopulation of GS-positive cells and that the GFAP-negative subpopulation is quite large (40%). After gliosis the density of GFAP-negative, GS-positive cells stays stable, whereas the GFAP-positive population triples. These experiments confirm electrophysiological experiments showing a distinct, GFAP-negative subset of astrocytes that remains consistent even after injury-induced gliosis and accompanying up-regulation of GFAP.
Over the past year, evidence has accrued that adult CNS stem cells are a widespread progenitor cell type. These cells may normally replace neurons and/or glia in the adult brain and spinal cord. Advances have been made in understanding the signals that regulate stem cell proliferation and differentiation. A deeper understanding of the structure of germinal zones has helped us move towards identifying stem cells in vivo. Recent studies suggest that the fate of stem cell progeny in vivo may be linked to the complexity of the animal's environment.
Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.
We describe a major glial cell population in the central nervous system (CNS) that can be identified by the expression of 2 cell surface molecules, the NG2 proteoglycan and the alpha receptor for platelet-derived growth factor (PDGF alphaR). In vitro and in the developing brain in vivo, NG2 and PDGF alphaR are expressed on oligodendrocyte progenitor cells but are down-regulated as the progenitor cells differentiate into mature oligodendrocytes. In the mature CNS, numerous NG2+/PDGF alphaR+ cells with extensive arborization of their cell processes are found ubiquitously long after oligodendrocytes are generated. NG2+ cells in the mature CNS do not express antigens specific to mature oligodendrocytes, astrocytes, microglia, or neurons, suggesting that they are a novel population of glial cells. Recently NG2+ cells in the adult CNS have been shown to undergo proliferation and morphological changes in response to a variety of stimuli, such as demyelination and inflammation, suggesting that they are dynamic cells capable of responding to changes in the environment. Furthermore, high levels of NG2+ and PDGF alphaR are expressed on oligodendroglioma cells, raising the possibility that the NG2+/PDGF alphaR+ cells in the mature CNS contribute to glial neoplasm.
Astrocytes are becoming increasingly recognized as targets for neurotoxic agents. For neurotoxicologists, as for other neuroscientists, an important concern is how to study astrocytes. In this paper, it is argued that studies of primary astrocyte cultures, while they are convenient as experimental systems and have been of great value to a resurgence of interest in these cell types over the past quarter century, now need to be supplemented to a large degree by studies on preparations where the properties of astrocytes are less likely to deviate from their properties in situ. Different and alternative systems to primary astrocyte cultures are described and critically evaluated in this article.
Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats. As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention. However, the actual classification of these electrophysiologically complex glial cells remains unclear. They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100beta, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP). To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats. In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells). We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA. We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive. We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively. In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP. The expression of GFAP at the transcript level indicates GFAP (-)/GFAP mRNA (+) glial cells have an astrocytic identity. As single cell RT-PCR is able to detect both GFAP (-)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.
Since 1992, it has been possible to record ionic currents from identified astrocytes in situ, using brain slice technology. Brain slice recordings confirm previous in vitro findings that expression of voltage-gated K(+) and Na(+) channels are a feature of this cell type. In contrast to cultured astrocytes, most investigators found that astrocytes in situ did not contain detectable, or at very best only low, levels of glial fibrillary acidic protein (GFAP). Structural and immunocytochemical investigations determined that these cells are different from oligodendrocyte precursors. In addition to cells with this current pattern, many but not all investigators found a second pool of astrocytes with no voltage-gated ion channels and high GFAP content. These two subpopulations of cells were termed complex and passive astrocytes. The existence of passive astrocytes has been questioned because of possible problems with space clamp conditions and spillage of EGTA-buffered pipette solution around the cells before recordings. Another problem is the fact there is a discrepancy regarding the GFAP content of complex astrocytes. It is of interest that recent immunocytochemical studies suggest the existence of two pools of astrocytes, one with a high GFAP content and one with nondetectable GFAP. Given this, it is tempting to correlate the two (controversial) electrophysiological patterns with immunochemical differences (GFAP) in order to demonstrate two functionally discrete classes of astrocytes in adult gray matter. However, despite evidence that some of the K(+) channels may be involved in proliferation, the role of voltage-gated ion channels in this nonexcitable cell type remains unknown. This is despite the fact that astrocytic Na(+) channels show dramatic changes after pathological events, re-enforcing the notion that the expression of this channel is under tight neuronal control. Several studies suggest that there is a great degree of flexibility and that astrocytes can undergo rapid changes in expression of both membrane ion currents and GFAP. Although it is likely that astrocytes exhibit different structural and membrane properties, this heterogeneity might be a reflection of the flexible plasticity of one astrocyte type under influence of environmental factors rather than of the existence of two distinct and permanent subtypes.
It is now well established that the glial fibrillary acidic protein (GFAP) is the principal 8-9 nm intermediate filament in mature astrocytes of the central nervous system (CNS). Over a decade ago, the value of GFAP as a prototype antigen in nervous tissue identification and as a standard marker for fundamental and applied research at an interdisciplinary level was recognized (Raine, 135). As a member of the cytoskeletal protein family, GFAP is thought to be important in modulating astrocyte motility and shape by providing structural stability to astrocytic processes. In the CNS of higher vertebrates, following injury, either as a result of trauma, disease, genetic disorders, or chemical insult, astrocytes become reactive and respond in a typical manner, termed astrogliosis. Astrogliosis is characterized by rapid synthesis of GFAP and is demonstrated by increase in protein content or by immunostaining with GFAP antibody. In addition to the major application of GFAP antisera for routine use in astrocyte identification in the CNS, the molecular cloning of the mouse gene in 1985 has opened a new and rich realm for GFAP studies. These include antisense, null mice, and numerous promoter studies. Studies showing that mice lacking GFAP are hypersensitive to cervical spinal cord injury caused by sudden acceleration of the head have provided more direct evidence for a structural role of GFAP. While the structural function of GFAP has become more acceptable, the use of GFAP antibodies and promoters continue to be valuable in studying CNS injury, disease, and development.
Caiman crocodilus, as a representative of the order Crocodilia, was used in immunohistochemical studies. Immunohistochemical procedures were performed on free-floating sections using a monoclonal antibody against porcine glial fibrillary acidic protein (GFAP) and employing standard avidin-biotin complex methodology. The astroglia of Caiman exhibited robust immunoreactivity to the antibodies raised against mammalian GFAP. In Caiman, the predominant GFAP-immunopositive elements are the radial ependymoglia, similar to other reptiles. The regional variability of glial architecture in Caiman, however, seems greater than in other reptiles so far examined, although it is less compared with chickens. We suggest that this finding corresponds to a more advanced "regional adaptation" of the glial structure in Caiman compared with other reptiles. The main feature that distinguishes the astroglia of Caiman from those of other reptiles is the widespread occurrence of GFAP-immunopositive astrocytes. These cells are limited in lizards and snakes, are not present in turtles, but are found in every major brain area in Caiman. However, even in Caiman, astrocytes are only intermingled with radial glia and are not the predominant glial element of any brain area. The occurrence of astrocytes does not correlate with brain wall thickness. Despite their origin from different ancestral groups of stem reptiles (synapsid or diapsid), mammals and birds exhibit some common general features in their glial architecture and GFAP distribution: 1) predominance of astrocytes and 2) absent or limited GFAP immunopositivity of several brain areas. The present study demonstrates that, even in Caiman, a representative of the reptilian group most closely related to birds, these features are present only in part, suggesting that, in mammals and birds, they have evolved independently.
The ability to control gene expression is central to normal development and function. For a growing number of genes in the central nervous system and peripheral tissues, expression is determined by changes in the rate of mRNA decay. At a molecular level, regulated interactions between the mRNA target and sequence-specific binding proteins either inhibit or accelerate decay, affording tight control over gene expression. This review discusses several examples of such posttranscriptional gene regulation.
Radial glia are specialized cells in the developing nervous system of all vertebrates, and are characterized by long radial processes. These processes facilitate the best known function of radial glia: guiding the radial migration of newborn neurons from the ventricular zone to the mantle regions. Recent data indicate further important roles for these cells as ubiquitous precursors that generate neurons and glia, and as key elements in patterning and region-specific differentiation of the CNS. Thus, from being regarded mainly as support cells, radial glia have emerged as multi-purpose cells involved in most aspects of brain development.
Neurogenesis continues into adult life in restricted germinal layers. The identification of the neural stem cells that give rise to these new neurons has important clinical implications and provides fundamental information to understand the origins of the new neurons. Work in adult birds and rodents yielded a surprising result: the neural stem cells appear to have characteristics of glia. In adult birds, the primary neuronal precursors are radial glia. In adult mammals, the primary neuronal precursors have properties of astrocytes. Radial glial cells have previously been shown to transform into astrocytes; both cell types are classically considered part of a committed astroglial lineage. Instead, we propose that neural stem cells are contained within this astroglial lineage. These findings in adult vertebrate brain, together with recent work in the developing mammalian cerebral cortex, force us to reexamine traditional concepts about the origin of neurons and glia in the central nervous system. In particular, neural stem cells possess a surprisingly elaborate structure, suggesting that in addition to their progenitor role, they have important structural and metabolic support functions. The very same cells that give birth to new neurons also seem to nurture their maturation and support their function.