Amin Derouiche

Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

Astroglial processes around identified glutamatergic synapses contain glutamine synthetase: evidence for transmitter degradation

Glutamate is the main excitatory transmitter in the cerebral cortex. The physiologically high spatial and temporal resolution in glutamatergic transmission requires effective transmitter removal. Thus, a close topochemical relation to the glutamatergic synapse is a prerequisite for an enzyme involved in glutamate transmitter degradation. Here we report that immunoreactivity against glutamine synthetase (GS), one of the glutamate metabolizing enzymes, is localized in the fine astrocytic processes associated with identified glutamatergic synapses in the rat hippocampus. We suggest that glutamate transmitter is rapidly taken up by these fine perisynaptic astrocytic processes and degraded by GS.

Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins.

Astrocytes extend thin lamellate processes in the neuropil, in particular around synapses, where they can modulate synaptic function or mediate glial–neuronal communication. Previous studies have shown that these lamellate perisynaptic processes change their shape in response to neuronal activity, but the underlying mechanisms have remained unclear. Similarly, the molecular composition of these fine, sheet‐like astrocytic processes (often 50–100 nm wide) is not understood but has to be related to their dynamic properties. To this end, we have studied the presence of ezrin, radixin, and moesin (ERM proteins) in the rat hippocampus and in primary cultured astrocytes, applying immunoperoxidase, immunofluorescence, and immunogold techniques. These three ERM proteins are known as actin‐binding proteins that link the cell membrane to the actin cytoskeleton, particularly in microvillus‐bearing epithelial cells. In cell culture, anti‐ezrin and antiradixin, but not antimoesin, antibodies were specific for astrocytes, which often displayed selective staining of filopodia and microvilli. Nonoverlapping visualization of astrocytic peripheral and stem processes was obtained by immunocytochemical double labeling for ezrin and GFAP, respectively. In sections of rat hippocampus, homogeneous labeling of the neuropil, but not of cell layers, resulted from immunostaining of fine, peripheral astrocyte processes, as confirmed ultrastructurally. Our data show that the fine peripheral processes of astrocytes, which also constitute the perisynaptic glial sheath, are specialized in that they contain characteristic actin‐associated molecules, likely to contribute to their dynamic properties. Applying anti‐ezrin and anti‐radixin as selective markers, plasticity of these perisynaptic glial processes can be analyzed. GLIA 36:330–341, 2001.

Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors

The peripheral astrocyte process (PAP) preferentially associates with the synapse. The PAP, which is not found around every synapse, extends to or withdraws from it in an activity-dependent manner. Although the pre- and postsynaptic elements have been described in great molecular detail, relatively little is known about the PAP because of its difficult access for electrophysiology or light microscopy, as they are smaller than microscopic resolution. We investigated possible stimuli and mechanisms of PAP plasticity. Immunocytochemistry on rat brain sections demonstrates that the actin-binding protein ezrin and the metabotropic glutamate receptors (mGluRs) 3 and 5 are compartmentalized to the PAP but not to the GFAP-containing stem process. Further experiments applying ezrin siRNA or dominant-negative ezrin in primary astrocytes indicate that filopodia formation and motility require ezrin in the membrane/cytoskeleton bound (i.e., T567-phosphorylated) form. Glial processes around synapses in situ consistently display this ezrin form. Possible motility stimuli of perisynaptic glial processes were studied in culture, based on their similarity with filopodia. Glutamate and glutamate analogues reveal that rapid (5 min), glutamate-induced filopodia motility is mediated by mGluRs 3 and 5. Ultrastructurally, these mGluR subtypes were also localized in astrocytes in the rat hippocampus, preferentially in their fine PAPs. In vivo, changes in glutamatergic circadian activity in the hamster suprachiasmatic nucleus are accompanied by changes of ezrin immunoreactivity in the suprachiasmatic nucleus, in line with transmitter-induced perisynaptic glial motility. The data suggest that (i) ezrin is required for the structural plasticity of PAPs and (ii) mGluRs can stimulate PAP plasticity.

Ezrin Immunoreactivity Is Associated with Increasing Malignancy of Astrocytic Tumors but Is Absent in Oligodendrogliomas

The actin-binding protein ezrin has been associated with motility and invasive behavior of malignant cells. To assess the presence of this protein in human glial cells of the brain and its potential role in benign and malignant glial tumors, we studied ezrin immunoreactivity (IR), proliferation (MIB-1-IR), and apoptosis (terminal dUTP nick-end labeling) in normal human brain tissues from 10 autopsies and tissues from 115 cases of human glial tumors including astro-cytomas, ependymomas, oligodendrogliomas, and glioblastomas. We found weak staining of peripheral processes in normal human brain astrocytes and in World Health Organization grade II benign astrocytomas. Staining was markedly increased in anaplastic astrocytomas (World Health Organization grade III) and clearly strongest in glioblastomas (World Health Organization grade IV). The increase of ezrin-IR correlated significantly with increasing malignancy of astrocytic tumors (P < 0.0001). Statistical analysis revealed a stronger association with increasing malignancy for ezrin-IR than for MIB-1-IR or terminal dUTP nick-end labeling staining. Ezrin-IR was absent in normal oligodendrocytes and in oligodendrogliomas, but pronounced in normal ependymal cells and ependymomas. Ezrin-IR seems to be specific for astrocytes and ependymal glia in the normal brain. Our results indicate that ezrin-IR may provide a useful tool for the distinction of oligodendrogliomas and astrocytomas and for the grading of astrocytic tumors.

Identified Glial Cells in the Early Postnatal Mouse Hippocampus Display Different Types of Ca2+ Currents

Based on their typical pattern of membrane currents, four populations of glial cells could be identified in thin brain slices of the postnatal hippocampus. In the present study, we applied the patch‐clamp technique to glial cells in the hippocampal CA1 region, which are characterized by a complex pattern of different Na+ and K− currents (“complex” cells). These cells were identified as non‐neuronal cells, most likely astrocytes, by their glutamine synthetase immunoreactivity. Two types of glial Ca2+currents could be identified that differed in their kinetics and pharmacological properties. A low‐voltage activated (LVA), fast inactivating component was activated at membrane potentials positive to −60 mV and reached maximum current amplitudes at about −20 mV. This current was sensitive to amiloride and thus displayed properties of neuronal LVA currents. The threshold potential of the second Ca2+ current component was at about −40 mV, and peak currents were observed at 0 mV. In contrast to the LVA component, the inactivation of these high‐voltage activated (HVA) currents slowed down with increasing depolarizations. This current was sensitive to low concentrations of Cd2+ but was not affected by amiloride. A small fraction of the HVA currents was sensitive to nifedipine, and ω‐conotoxin GVIA (ω‐CgTx) was also found to reduce the glial HVA component. The study provides electrophysiological and pharmacological characterization of different types of Ca2+ currents in gray matter glial cells in situ.

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

BackgroundThe 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 Sonic Hedgehog (Shh). However, Shh 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 Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points.ResultsWe employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (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 Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes.ConclusionsWe define a ventral midbrain precursor map based on the timing of Gli1 and Shh 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.

The dopamine D2 receptor subfamily in rat retina: ultrastructural immunogold and in situ hybridization studies.

Dopamine, a major neurotransmitter in the vertebrate retina, is released from interplexiform cells and a restricted subset of amacrine cells. Dopamine effects vary between different retinal cell types, most likely due to differences in cell‐specific receptor subtype expression. Identification of cells expressing receptors of the D2‐subfamily (D2R, D3R, D4R) on a light microscopical level has rendered equivocal results, and no information is as yet available concerning the subcellular distribution of receptor protein. In the present study, D2R and D2/3R subtype‐specific antisera, and D2R‐, D3R‐ and D4R‐specific oligonucleotide probes were used for ultrastructural and in situ hybridization analyses of the receptor subtype distribution in the rat retina.

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.

An immunocytochemical investigation of glial morphology in the Pacific hagfish: radial and astrocyte-like glia have the same phylogenetic age

SummaryThis study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish,Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-pfotein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.