Keiko Yamada

Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST‐deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord.

The glutamate transporter GLAST is localized on the cell membrane of mature astrocytes and is also expressed in the ventricular zone of developing brains. To characterize and follow the GLAST-expressing cells during development, we examined the mouse spinal cord byin situ hybridization and immunohistochemistry. At embryonic day (E) 11 and E13, cells expressing GLAST mRNA were present only in the ventricular zone, where GLAST immunoreactivity was associated with most of the cell bodies of neuroepithelial cells. In addition, GLAST immunoreactivity was detected in radial processes running through the mantle and marginal zones. From this characteristic cytology, GLAST-expressing cells at early stages were judged to be radial glia cells. At E15, cells expressing GLAST mRNA first appeared in the mantle zone, and GLAST-immunopositive punctate or reticular protrusions were formed along the radial processes. From E18 to postnatal day (P) 7, GLAST mRNA or its immunoreactivity gradually decreased from the ventricular zone and disappeared from radial processes, whereas cells with GLAST mRNA spread all over the mantle zone and GLAST-immunopositive punctate/reticular protrusions predominated in the neuropils. At P7, GLAST-expressing cells were immunopositive for glial fibrillary acidic protein, an intermediate filament specific to astrocytes. Therefore, the glutamate transporter GLAST is expressed from radial glia through astrocytes during spinal cord development. Furthermore, the distinct changes in the cell position and morphology suggest that both the migration and transformation of radial glia cells begin in the spinal cord between E13 and E15, when the active stage of neuronal migration is over.

Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells

Bergmann glia (BG) are unipolar cerebellar astrocytes, whose radial (or Bergmann) fibers associate with developing granule cells and mature Purkinje cells (PCs). In the present study, we investigated the morphodifferentiation of BG by immunohistochemistry for glutamate transporter GLAST and electron microscopy. GLAST was expressed widely in cerebellar radial glia/astrocytes during fetal and neonatal periods and became concentrated in BG postnatally. During the second postnatal week when PC dendrites grow actively, GLAST immunostaining revealed dynamic cytologic changes in Bergmann fibers in a deep‐to‐superficial gradient; Bergmann fibers traversing the external granular layer were stained as rod‐like fibers, whereas in the molecular layer, the rod‐like pattern was gradually replaced with a reticular meshwork. At postnatal day 10, the superficial rod‐like domain was composed of glial fibrillary acidic protein (GFAP)‐positive/GLAST‐positive straight fibers, forming cytoplasmic swellings and short filopodia. Along this domain, the tip of growing PC dendrites ascended vertically and entered the base of the external granular layer. The deeper reticular domain of Bergmann fibers was characterized by active expansion of GFAP‐negative/GLAST‐positive lamellate processes, which surrounded PC synapses almost completely. Therefore, the transformation of Bergmann fibers proceeds in correlation with dendritic differentiation of PCs. The intimate PC‐BG relationships during cerebellar development raise the possibility that a preexisting glial shaft could serve as a structural substrate that directs dendritic outgrowth toward the pial surface, whereas the successive formation of a reticular glial meshwork should lead to structural maturation of newly formed PC synapses. J. Comp. Neurol. 418:106–120, 2000.

EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses.

To study cellular and subcellular localizations of the glutamate transporter EAAT4, antibody was raised against the N-terminal peptide. On immunoblotting the antibody recognized a band in membrane extracts from the cerebellum, but not from the forebrain. Immunohistochemistry revealed that its distribution was restricted to the cerebellar molecular layer, where the immunoreactivity was observed as numerous punctate stainings. Electron microscopy showed the antibody to label dendritic spines of the Purkinje cells. EAAT4 is, therefore, a Purkinje cell-specific, postsynaptic transporter. Together with dense localization of other transporter subtypes in Bergmann astrocytic membranes, Purkinje cell synapses are thus provided with distinct glutamate transporter subtypes at discrete synaptic elements, which would play important roles in regulating excitability of the Purkinje cells and protecting against excitotoxicity.

Glutamate Transporter GLT-1 Is Transiently Localized on Growing Axons of the Mouse Spinal Cord before Establishing Astrocytic Expression

The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, 35S-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.

Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells

EAAT4 is a Purkinje cell (PC)-specific, postsynaptic glutamate transporter in the adult mouse brain. Here, we performed developmental analyses to reveal its temporal expression in relation to PC differentiation. Using in situ hybridization, EAAT4 mRNA was specifically detected in the PC layer of the cerebellar primordium at embryonic day 13 (E13). During late fetal and neonatal periods, the transcripts were detected only in the PC layer in the caudal cerebellum. At postnatal day 7 (P7) and, thereafter, the prominent transcripts were found on monolayered PCs in the entire cerebellum. Using immunohistochemistry, low levels of EAAT4 immunoreactivity were first observed at E18 in the perikarya of PCs in the caudal cerebellum, and this pattern of immunostaining was maintained at P1 and P7. At P14 and, thereafter, the molecular layer in the entire cerebellum became immunopositive for EAAT4, and the intense immunoreactivity was detected preferentially in PC spines synapsing with parallel fiber terminals. Therefore, the present study has clarified that the transcription of EAAT4 begins in PCs from early embryonic stages, and that the synaptic localization of EAAT4 is established during the second postnatal week. When considered in the light of the synaptogenesis of parallel fiber-PC synapses which actively occurs in the rodent cerebellum during the second and third weeks of life, synaptic localization of the glutamate transporter EAAT4 may be closely associated with the synapse formation.

DOWN-REGULATED EXPRESSION OF GLUTAMATE TRANSPORTER GLAST IN PURKINJE CELL-ASSOCIATED ASTROCYTES OF REELER AND WEAVER MUTANT CEREBELLA

The glutamate transporter plays an important role in rapid removal of glutamate from the synaptic cleft. Glutamate transporter GLAST is highly expressed in the Bergmann glia (BG), a unipolar cerebellar astrocyte associated structurally and functionally with Purkinje cells (PCs). Here we investigated the expression and localization in the reeler and weaver mutant cerebella with disorganized cytoarchitecture and disrupted synaptic circuitry. In the cortex of both cerebella, GLAST-expressing cells were astrocytes associating PCs; they were located around PC somata and primary dendrites, and extended glial fibrillary acidic protein (GFAP)-immunopositive processes surrounding PC somata and dendrites. Additional signals were detected in astrocytes of the reeler subcortex; they were dispersed among ectopic PCs and had GFAP-positive processes apposing to PC somata and stunted dendrites. Therefore, GLAST expression in PC-associated astrocytes was conserved in these mutants. Compared to the wild-type BG, however, the transcription level in individual mutant astrocytes was significantly reduced to about one-third level in the reeler and weaver cortex or one-sixth level in the reeler subcortex. Taking previous results on remarkable up-regulation during dendritogenic/synaptogenic stages and down-regulation following experimental glutamatergic denervation, it is suggested that GLAST expression in cerebellar astrocytes is regulated correlatively with cytological and/or synaptic differentiation of neighboring PCs.

Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice

The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy.