Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)

Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13–15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5–3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated

FTY720 treatment in the convalescence period improves functional recovery and reduces reactive astrogliosis in photothrombotic stroke

Background: The Sphingosine-1-phosphate (S1P) signaling pathway is known to influence pathophysiological processes within the brain and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P signaling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. Methods: We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence. Results: FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in reactive astrogliosis and larger post-synaptic densities as well as changes in the expression of vascular endothelial growth factor α (VEGF α). Within the periinfarct cortex, S1P is significantly increased compared to healthy brain tissue. Conclusion: Besides its known neuroprotective effects in the acute phase of experimental stroke, the initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors

Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

<p>Abstract</p> <p>Background</p> <p>The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express <it>Sonic Hedgehog </it>(<it>Shh</it>). However, <it>Shh </it>expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene <it>Gli1 </it>initiates in the ventral midline prior to <it>Shh </it>expression, but after the onset of <it>Shh </it>expression it is expressed in precursors lateral to <it>Shh</it>-positive cells. Given these dynamic gene expression patterns, <it>Shh </it>and <it>Gli1 </it>expression could delineate different progenitor populations at distinct embryonic time points.</p> <p>Results</p> <p>We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express <it>Shh </it>(Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from <it>Shh</it>- and <it>Gli1</it>-expressing progenitors located outside of the ventral midline give rise to astrocytes.</p> <p>Conclusions</p> <p>We define a ventral midbrain precursor map based on the timing of <it>Gli1 </it>and <it>Shh </it>expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.</p

Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain

While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain

Perspectives for Ezrin and Radixin in Astrocytes: Kinases, Functions and Pathology

Astrocytes are increasingly perceived as active partners in physiological brain function and behaviour. The structural correlations of the glia–synaptic interaction are the peripheral astrocyte processes (PAPs), where ezrin and radixin, the two astrocytic members of the ezrin-radixin-moesin (ERM) family of proteins are preferentially localised. While the molecular mechanisms of ERM (in)activation appear universal, at least in mammalian cells, and have been studied in great detail, the actual ezrin and radixin kinases, phosphatases and binding partners appear cell type specific and may be multiplexed within a cell. In astrocytes, ezrin is involved in process motility, which can be stimulated by the neurotransmitter glutamate, through activation of the glial metabotropic glutamate receptors (mGluRs) 3 or 5. However, it has remained open how this mGluR stimulus is transduced to ezrin activation. Knowing upstream signals of ezrin activation, ezrin kinase(s), and membrane-bound binding partners of ezrin in astrocytes might open new approaches to the glial role in brain function. Ezrin has also been implicated in invasive behaviour of astrocytomas, and glial activation. Here, we review data pertaining to potential molecular interaction partners of ezrin in astrocytes, with a focus on PKC and GRK2, and in gliomas and other diseases, to stimulate further research on their potential roles in glia-synaptic physiology and pathology

Glutamine synthetase as an astrocytic marker: its cell type and vesicle localization

The overall staining by GS clearly reveals astrocytes, including all cells of the astroglial family (1), i.e., Bergmann glia, Müller cells (2), tanycytes (3), and ependymal cells. The star shaped morphology from classical silver impregnations relates to cortical and hippocampal astrocytes, which display a comparable pattern in material stained for GFAP. However, the dense population of GS stained astrocytes found in all diencephalic and mesencephalic regions, known to display faint GFAP-labeling (unpublished observations) indicated that while apparently all astrocytes contain GS they have GFAP-ir filaments only in a region-dependent pattern. This is complicated by the emerging view that "astrocytes" constitute a heterogeneous population even within a given region. In the rat hippocampus, combined immunostainings have revealed that the "classical" GFAP-ir astrocyte constitutes a subpopulation of GS-ir astrocytes, which can also lack GFAP staining [direct double staining (4)]. In view of several astroglial subtypes and/or glial precursors present in the adult rodent brain, anti-GS appears to be the most general astrocyte marker, covering all subtypes. In addition, GS has been found early on to label exclusively astrocytic cells and no other glial or neuronal cell types in situ or in culture [reviewed by (5)]. GS has, thus, been applied as a reliable astrocyte marker in very many studies since. "Complex cells" in rat hippocampus, initially assumed to be an astrocyte subtype (6) but now understood to belong to NG2 cells, a fourth glial type in the CNS (7), may display faint GS-ir in the soma but not its fine processes. Oligodendrocyte precursor cells, possibly also related to NG2 cells, were found to be devoid of GS-ir (8). The immunocytochemical profile and possible heterogeneity of NG2 cells is still under debate to date. Disputing the exclusion of non-astrocytic cells in GS staining, some authors have later reported GS+ oligodendrocytes, although this has not been investigated systematically. Reports on non-astrocytic GS will be discussed in detail here

Perspectives for ezrin and radixin in astrocytes: kinases, functions and pathology

Astrocytes are increasingly perceived as active partners in physiological brain function and behaviour. The structural correlations of the glia–synaptic interaction are the peripheral astrocyte processes (PAPs), where ezrin and radixin, the two astrocytic members of the ezrin-radixin-moesin (ERM) family of proteins are preferentially localised. While the molecular mechanisms of ERM (in)activation appear universal, at least in mammalian cells, and have been studied in great detail, the actual ezrin and radixin kinases, phosphatases and binding partners appear cell type specific and may be multiplexed within a cell. In astrocytes, ezrin is involved in process motility, which can be stimulated by the neurotransmitter glutamate, through activation of the glial metabotropic glutamate receptors (mGluRs) 3 or 5. However, it has remained open how this mGluR stimulus is transduced to ezrin activation. Knowing upstream signals of ezrin activation, ezrin kinase(s), and membrane-bound binding partners of ezrin in astrocytes might open new approaches to the glial role in brain function. Ezrin has also been implicated in invasive behaviour of astrocytomas, and glial activation. Here, we review data pertaining to potential molecular interaction partners of ezrin in astrocytes, with a focus on PKC and GRK2, and in gliomas and other diseases, to stimulate further research on their potential roles in glia-synaptic physiology and pathology

Rab6A as a pan-astrocytic marker in mouse and human brain, and comparison with other glial markers (GFAP, GS, Aldh1L1, SOX9)

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