Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells
Introduction
Glutamate is a major neurotransmitter involved in fast excitatory synaptic transmission in the mammalian central nervous system (Mayer and Westbrook, 1987), and is essential for synaptic plasticity, which is thought to underlie development, learning, and memory (Ito, 1989; McDonald and Johnston, 1990; Bliss and Collingridge, 1993). Glutamate released from presynaptic terminals has to be removed rapidly from the synaptic cleft by high affinity, sodium-dependent glutamate transporters to keep the extracellular glutamate concentration sufficiently low to terminate receptor activation and to protect neurons from glutamate excitotoxicity (Hertz, 1979; Choi, 1992). Recent studies have identified four subtypes of the glutamate transporter with distinct structures, functions, and expression patterns: GLAST (GluT-1, EAAT1), GLT1 (EAAT2), EAAC1 (EAAT3), and EAAT4 (Storck et al., 1992; Kanai and Hediger, 1992; Kanai et al., 1993; Kanner, 1993; Tanaka, 1993; Torp et al., 1992; Rothstein et al., 1994; Fairman et al., 1995; Kanai et al., 1995; Lehre et al., 1995; Shibata et al., 1996; Yamada et al., 1996).
Purkinje cells (PCs) in the cerebellum receive massive glutamatergic inputs from parallel fibers, the bifurcated axons of granule cells (Palay and Chan-Palay, 1974; Ito, 1989). The parallel fiber-PC synapse is estimated to be 105 per PC (Napper and Harvey, 1988), and formed on PC spines of distal dendrites. In addition, climbing fibers from the inferior olive establish strong excitatory synapses along the proximal dendrites, which trigger Ca2+ entry through voltage-gated Ca2+ channels (Sakurai, 1990; Hirano, 1990; Crepel and Jaillard, 1991; Konnerth et al., 1992). Immunohistochemical studies have revealed that several subtypes of the glutamate transporter are localized at discrete elements of PC synapses. GLAST and GLT1 are localized densely on the cell membrane of the Bergmann astroglia, which seals the PC synapses (Rothstein et al., 1994; Lehre et al., 1995). EAAT4, on the other hand, is localized selectively in dendritic spines of PCs (Yamada et al., 1996). In view of the importance of rapid glutamate clearance in regulating synaptic transmission and in protecting neurons from glutamate excitotoxicity, the formation of glutamatergic synapses should be accompanied by the establishment of the glutamate transporter system. In fact, recent expression studies have shown that transcription of GLAST and GLT1 undergoes marked up-regulation during early postnatal periods (Shibata et al., 1996; Sutherland et al., 1996). To clarify developmental regulation of the gene expression for EAAT4 in relation to the differentiation and synaptogenesis of the PCs, we performed analyses in developing mouse cerebella using in situ hybridization and immunohistochemistry.
Section snippets
In situ hybridization
Two non-overlapping antisense oligonucleotides were used for detection of the mouse EAAT4 mRNA in the present study. The sequences of the oligonucleotides are 5′- GCCCCCAGCTCTGAACCATTGTCTGTCCTTACAATTGTCCTTGTCA-3′ and 5′-GATGCCCCCTTTTCTTGTGCCATGAGTGACTTATAGGGTTTCCCCA-3′, which are complementary to nucleotide residues 611–656 (probe 1) and 1607–1652 (probe 2), respectively, of the mouse EAAT4 cDNA (Maeno-Hikichi et al., unpublished data). These oligonucleotides were labeled with [33P]dATP to a
In situ hybridization
Using in situ hybridization with 33P-radiolabeled oligonucleotide probes, expression of EAAT4 mRNA was examined in developing mouse brains from E13 to P120 (Fig. 1Fig. 2). EAAT4 mRNA was expressed in the cerebellum at E13, when the transcripts were observed as a thin layer running parallel to the roof of the fourth ventricle (Fig. 1A). At E15, EAAT4 mRNA was concentrated in the mantle zone of the dorsal cerebellum (Fig. 1B and Fig. 2A). At this stage, cells expressing EAAT4 mRNA were virtually
Discussion
EAAT4 is a member of the family of glutamate transporters, which play an important role in rapid clearing of extracellular glutamate (Kanai and Hediger, 1992; Pines et al., 1992; Storck et al., 1992; Tanaka, 1993; Fairman et al., 1995). From the selective expression and localization, we have previously concluded that EAAT4 is a PC-specific, postsynaptic transporter in the adult mouse brain (Yamada et al., 1996). In the present study, we performed developmental analyses, and elucidated
Acknowledgements
We thank Hideo Umeda and Yoshihiko Ogawa for their technical assistance. This investigation was supported in part by research grants from the Ministry of Education, Science, Sports and Culture, the Ministry of Health and Welfare and the Science and Technology Agency of Japan.
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