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. 2004 Feb 4;24(5):1136-48.
doi: 10.1523/JNEUROSCI.1586-03.2004.

The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

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Free PMC article

The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

Weizhi Chen et al. J Neurosci. .
Free PMC article

Abstract

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3'-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14-29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

Figures

Figure 1.
Figure 1.
In situ hybridization analysis of the distribution of mRNAs for full-length GLT1, GLT1a, and GLT1b in the hippocampus. GLT1 mRNA was labeled in hippocampus using probes covering the full-length GLT1 coding sequence (pan-GLT1 probe) (A, D, E, J, M, N) or specific stretches of the 3′-UTR sequence of GLT1a (B, F, G, K, O, P) and GLT1b (C, H, I, L, Q, R). The top two rows (A–I) illustrate single in situ hybridization labeling obtained with AP detection. The bottom two rows (J–R) show double hybridized sections in which the fluorescent labeling of astrocytic mRNA is blocked or attenuated, because of quenching by the AP reaction product associated with a GLAST probe. The second (D–I) and fourth (M–R) rows show magnified views of the CA1 (left panel; D, F, H, M, O, Q) and CA3 (right panel; E, G, I, N, P, R) subfields of the hippocampi shown in the row above, as outlined by the boxes shown in A. A, D, E, The pan-GLT1 probe produces prominent neuronal labeling for GLT1 in pyramidal cell neurons in the CA3 (E) subfield but not in CA1 (D). Astrocytes throughout the hippocampus and dentate gyrus are also labeled. B, F, G, GLT1a labeling shows a similar pattern of neuronal labeling, although at reduced intensity. Astrocytic labeling is present but reduced compared with that in A. C, H, I, GLT1b labeling is prominent in astrocytes throughout the hippocampus. CA3 neurons are not clearly labeled (I). J, M, N, Fluorescent labeling using the pan-GLT1 probe highlights the strong labeling of CA3 neurons (J, N). Astrocytic labeling in the CA1 region (M) is blocked by the AP deposit produced by the cohybridized GLAST probe, which only labels astrocytes. K, O, P, Strong GLT1a fluorescent labeling in CA3 neurons (K, P) but not in CA1 neurons (O). L, Q, R, The differential double-in situ technique reveals weak, but distinct, GLT1b fluorescent labeling in the CA3 region (R and arrows in L) but not in CA1(Q). Note that the photographic exposure time was longer for L, Q, and R compared with J, K, and M-P because of the much weaker signal obtained with the GLT1b probe. The lower signal/noise ratio with the GLT1b probe is evident based on the more apparent background labeling in L, Q, and R. The labeling by the GLT1b probe is locally very restricted, because of the low abundance of the message and the amplification technique that gains sensitivity at the expense of resolution. The area of apparent labeling under the arrow shafts in L is attributable to a bubble under the section. These findings demonstrate that the GLT1a isoform is expressed in CA3 neurons. The GLT1b transcript is weakly expressed in the CA3 region. The transcript for GLT1a appears to be predominant in neurons. Bars: L, 500 μm; R, 100 μm.
Figure 2.
Figure 2.
High magnification analysis of the GLT1b labeling in CA3 neurons with DAPI nuclear counterstaining. Sections processed for GLT1b (A, B, E, F) and GLAST (C, D, G, H) AP labeling were coincubated with the nuclear counterstain DAPI to visualize the neuronal nuclei. The pictures show the AP labeling simultaneously with the fluorescent DAPI staining. At low magnification, no GLT1b labeling is present in the CA1 neurons (A, arrows), but a faint labeling can be discerned in CA3 neurons (B, arrows). In contrast, the GLAST labeling is restricted to astrocytes and is not present in CA1 or CA3 neurons (C, D), suggesting the GLT1b labeling in the CA3 neurons is specific. At high magnification, it becomes apparent that the GLT1b labeling is localized to a small rim of cytoplasm surrounding the nucleus in CA3 neurons (F, arrows) and that the GLT1b message is not present in CA1 neurons (E). Similarly, the GLAST message is not present in the CA1 or CA3 neurons (G, H). The GLAST profiles labeled within the pyramidal cell layer are astrocyte processes that surround the neurons. Bars: D, 100 μm; H, 10 μm.
Figure 3.
Figure 3.
Comparison of anti-cGLT1a antibody immunoreactivity in immunoblot assay of brain lysates from littermates of a GLT1+/– × GLT1+/– mating. A, Characterization of genotypes was achieved by amplification of the cDNAs encoding for exon 4 and the neomycin resistance gene sequence using PCR. The PCR products were run on a 1% agarose gel and stained with ethidium bromide. The 243 bp band indicates the presence of the wild-type exon 4 sequence, whereas the 341-bp band indicates the presence of the neomycin resistance gene sequence that replaces the exon 4 sequence in the GLT1 knock-out mouse. B, Immunoreactivity of the anti-cGLT1a, anti-cGLT1b, and anti-nGLT1 antibodies in brain lysates was completely abolished in the GLT1 knock-out mouse. Brain lysates were subjected to SDS-PAGE, immunoblotted, and detected with the anti-cGLT1a, anti-cGLT1b, and anti-nGLT1 antibodies. C, Equal loading of proteins on the blots was verified by staining of membrane with Ponceau S. GLT1+/+, Wild-type GLT1 mouse; GLT1+/–, heterozygote GLT1 mouse; GLT1–/–, homozygote GLT1 knock-out mouse.
Figure 4.
Figure 4.
Comparison of immunoreactivity of anti-cGLT1a and anti-nGLT1 antibodies in the hippocampus of wild-type and GLT1–/– knock-out mice at P7 LM visualization of GLT1 immunoreactivity in the CA3 region of the hippocampus of wild-type and GLT1 knock-out mice. AD show micrographs taken from tissue stained using the anti-cGLT1a antibody, whereas EH show tissue stained using the anti-nGLT1 antibody. A, C, E, and G show micrographs taken at a magnification of 10× (calibration, 200 μm). Strata of the hippocampus: o, stratum oriens; p, stratum pyramidale; r, stratum radiatum; the dashed lines indicate the transition of the strata. Details of the boxed portion are shown in adjacent panels using micrographs taken at a magnification of 40× (calibration, 50 μm).
Figure 5.
Figure 5.
LM visualization of GLT1 immunoreactivity within the adult rat hippocampus. The two columns of panels show immunoreactivity of the anti-cGLT1a- and nGLT1 antibodies, respectively, using HRP–DAB as labels. The light micrographs shown in A, C, D, and F were taken at a magnification of 10×. The large arrows point to the CA1/CA3 transition. Within the two fields of both anti-cGLT1a and anti-nGLT1-stained tissue, the unlabeled band coincides with the pyramidal cell layer. The granule cell layer in the dentate gyrus also is minimally immunoreactive. Viewing of the CA1/CA3 transition at a higher magnification of 40× (B, E, middle) reveals absence of labeling within perikarya and apical dendrites of these pyramidal neurons but, additionally, small puncta distributed throughout the neuropil dorsal and ventral to the cell body layer. C and F show the preadsorption control, in which the synthetic peptide used to generate the antisera was added before tissue incubation, resulting in a nearly complete abolishment of immunoreactivity. Scale bar: top and bottom, 400 μm; middle, 100 μm.
Figure 6.
Figure 6.
Electron microscopy reveals GLT1a immunoreactivity specifically in axon terminals, astrocytes, and dendrites. Immunoreactivity is visualized by the flocculent, electron-dense, peroxidase reaction product along membranes. A and B show examples of immunoreactive terminals (T) forming asymmetric axo-spinous juctions. Here and in other panels, the large open arrows point to PSDs, whereas small arrows point to portions of neurons exhibiting high concentrations of immunoreactivity. C shows the specificity of labeling of axon terminals and astrocytic processes within a single section. Immunocytochemical conditions that produced astrocytic labeling, as reported previously, also resulted in detection of GLT1a within some, but not all, axo-spinous excitatory junctions, and only rarely within dendrites. The field shown in C contains four axo-spinous synapses, two of which exhibit immunoreactivity within the axon terminal. The other two synapses show no detectable levels of immunoreactivity within the terminals (UT, unlabeled terminal). In the same field, astrocytic processes surround the dendritic shaft portions of the spine receiving synaptic input from the labeled terminal. These astrocytic processes exhibit immunoreactivity along the intracellular surface (arrowheads). D shows an example of a dendritic shaft exhibiting immunoreactivity along the smooth endoplasmic reticulum near a PSD. E shows complete absence of immunoreactivity in terminals, astrocytes (*), and spines after preadsorption of the antibody with the synthetic peptide against which the antibody was generated. Scale bars: A, C–E, 500 nm; B, 625 nm.
Figure 7.
Figure 7.
Anti-nGLT1 antibody also labels axons and spines. In all panels, immunoreactivity is indicated by thin arrows in neurons and arrowheads in astrocytes. The large, open arrows point to PSDs within spines. A shows nGLT immunoreactivity along the plasma membrane of an axon (Ax) forming a synapse onto a spine. The terminal portion of that axon is unlabeled. The shaft portion of the same dendrite exhibits immunoreactivity along the plasma membrane. Within the same field, immunoreactivity along the plasma membrane of an astrocytic process is also shown. B shows immunoreactivity along the plasma membrane of a dendritic spine and an astrocyte in its vicinity. C shows immunoreactivity in two axon terminals (T) in the vicinity of an unlabeled terminal (UT). D shows complete abolishment of immunoreactivity within the synaptic neuropil, including astrocytic processes (*), after preadsorption of the antibody with the peptide used to generate it. Scale bar, 500 nm.
Figure 8.
Figure 8.
Subcellular distribution of immunoreactivity using the anti-cGLT1a and anti-nGLT1 antibodies. A minimum of 10 photographs for each hippocampal region were taken of sections from two animals for each antibody and were examined at magnifications of 12,000×, 15,000×, or 20,000×. The total number of labeled profiles encountered were 1393 for the anti-GLT1a antibody-1abeled profiles and 1096 for the anti-nGLT1 antibody-labeled profiles. After excluding morphologically unidentifiable profiles, the percentages of all immunolabeled profiles from each micrograph that were astrocytes, axons, spines, or shafts were determined. The graphs show mean and SEM values in percentages of all morphologically identifiable, immunolabeled profiles encountered in each micrograph representing each region and each antibody. Astrocytic profiles were the major subcellular profiles immunolabeled. Among the neuronal profiles, axons were immunolabeled more frequently than spines (unpaired t test; *p ≤ 0.05). N values (the number of micrographs sampled) were the following for the cGLT1a antibody: 50 from the CA1 region, 22 from CA3 and 28 from DG. The N values for the nGLT1 analysis were 31 for CA1 region, 33 for CA3 and 35 for DG.
Figure 9.
Figure 9.
Comparison of axonal labeling across regions of the hippocampus. The same set of electron micrographs used for the analyses shown in Figure 8 was used here to determine the proportion of all axons encountered that were immunolabeled. The graphs show mean ± SEM values. To test for differences among the CA1, CA3, and DG groups, ANOVA was performed. Anti-cGLT1a antibody immunoreactivity in axons was heterogeneous, and the difference in axon labeling between the CA1 and CA3 fields was significant (p = 0.016). The difference in axon labeling across regions using the anti-nGLT1 antibody was not statistically significant. The N values for the anatomical regions and antibodies were the same as indicated in the legend to Figure 8.
Figure 10.
Figure 10.
GLT1a immunoreactivity within hippocampal terminals and astrocytes of P7 mice. The tissue was obtained from +/+ (A, B) and –/– (C) littermates of a GLT1+/– × GLT1+/– cross. A shows two profiles immunolabeled with the anti-cGLT1a antibody, identifiable as terminals (T) by the presence of a few vesicles (small arrows). Both A and B show immunolabeled profiles identifiable as astrocytes by the irregular contours of their plasma membranes (arrowheads). Both images were taken from the stratum radiatum of the CA1. In A, a putative postsynaptic specialization is marked with an open arrow. The LM results from the same tissue are shown in Figure 4, which also shows images obtained from age-matched knock-out littermates. C shows GLT1a immunoreactivity in a knock-out (–/–) littermate. The tissue was sampled from the CA3 region of the hippocampus. White arrowheads point to the tissue surface in which exposure to immunoreagents is expected to be the greatest and demonstrate that this section was, in fact, taken from this region of the block. Scale bar (in B): A, B, 500 nm; C, 500 nm.

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