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      Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells


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          Here we show that GLAST, a major glutamate transporter in the cerebellar cortex, is essential for synaptic wrapping by Bergmann glia and synaptic wiring on Purkinje cells (PCs) by parallel fibers (PFs) and climbing fibers (CFs). Without GLAST, monoinnervation of PCs by single strong CFs and segregation of CF and PF territories along PC dendrites cannot develop normally or be maintained. PCs are frequently innervated by additional CF, whereas innervation by main CFs becomes weaker. Ectopic PF synapses appear at proximal dendrites, causing disruption of CF and PF territory segregation along PC dendrites. We conclude that GLAST is indispensable for the establishment of excitatory synaptic wiring to PCs through competition between CFs and between CFs and PFs.


          Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/ l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF–PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10–90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.

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          Most cited references44

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          Localization of neuronal and glial glutamate transporters.

          The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.
            • Record: found
            • Abstract: found
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            Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells.

            Astrocytes are considered a reticulate network of cells, through which calcium signals can spread easily. In Bergmann glia, astrocytic cells of the cerebellum, we identified subcellular compartments termed 'glial microdomains'. These elements have a complex surface consisting of thin membrane sheets, contain few mitochondria and wrap around synapses. To test for neuronal interaction with these structures, we electrically stimulated parallel fibers. This stimulation increased intracellular calcium concentration ([Ca2+]i) in small compartments within Bergmann glial cell processes similar in size to glial microdomains. Thus, a Bergmann glial cell may consist of hundreds of independent compartments capable of autonomous interactions with the particular group of synapses that they ensheath.
              • Record: found
              • Abstract: found
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              Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry.

              The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer approximately hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerve terminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsin on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.

                Author and article information

                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                11 July 2017
                27 June 2017
                : 114
                : 28
                : 7438-7443
                [1] aDepartment of Anatomy, Hokkaido University Graduate School of Medicine , Sapporo 060-8638, Japan;
                [2] bDepartment of Neurophysiology, Graduate School of Medicine, The University of Tokyo , Hongo, Tokyo 113-0033, Japan;
                [3] cDepartment of Neurophysiology, Graduate School of Biomedical and Health Sciences, Hiroshima University , Hiroshima 734-8551, Japan;
                [4] dDepartment of Physiology, Keio University School of Medicine , Tokyo 160-8582, Japan;
                [5] eDepartment of Physiology, St. Marianna University School of Medicine , Kanagawa 216-8511, Japan;
                [6] f Bioorganic Research Institute, Suntory Foundation for Life Sciences , Kyoto, 619-0284, Japan;
                [7] gLaboratory of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University , Tokyo 113-8510, Japan
                Author notes
                1To whom correspondence should be addressed. Email: watamasa@ 123456med.hokudai.ac.jp .

                Edited by Tomas G. M. Hokfelt, Karolinska Institutet, Stockholm, Sweden, and approved June 1, 2017 (received for review October 18, 2016)

                Author contributions: T.M. and M.W. designed research; T.M. and M. Yamasaki performed research; K.K., M. Yuzaki, and K.S. contributed new reagents/analytic tools; T.M. and M. Yamasaki analyzed data; K.T. and M.K. supervised electrophysiology; and T.M., M. Yamasaki, K.H., K.T., M.K., and M.W. wrote the paper.

                PMC5514701 PMC5514701 5514701 201617330
                Page count
                Pages: 6
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 24220007
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 25000015
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 26460250
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 26460251
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 25117006
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 16H01615
                Funded by: Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 501100001700
                Award ID: 17H03551
                Biological Sciences

                Bergmann glia,parallel fiber,climbing fiber,Purkinje cell,glutamate transporter


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