Localization of neuronal and glial glutamate transporters

Neuron,

Vol. 13, 713-725, September,

1994, Copyright

0 1994 by Cell Press

localization of Neuronal and Clial G lutamate Transporters Jeffrey D. Rothstein,* lee Martin,+ Allan I. Levey,§ Margaret Dykes-Hoberg,* Lin Jin,* David Wu,* Norman Nash) Ralph W. Kuncl*t *Department of Neurology tDepartments of Neuroscience and Pathology and The Neuropathology Laboratory *Graduate Program in Cellular and Molecular Biology The Johns Hopkins University Baltimore, Maryland 21287-7519 SDepartment of Neurology Emory University School of Medicine Atlanta, Georgia 30322 IIScios-Nova 6200 Freeport Centre Baltimore, Maryland 21224-6522

Summary The cellular and subcellular distributions of the glutamate transporter subtypes EAACl, CLT-1, and CIAST 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. lmmunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAACl 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. GLUT is found in both neurons and astroglia. The regional localizations are uniquetwach transporter subtype. EAACl is highly enriched in the cortex, hippo campus, and caudate-putamen and is confined to preand postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLUT is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei. Introduction Synaptic transmission is largely terminated by high affinity, sodium-dependent transport of neurotransmitters from the synaptic cleft (Iversen, 1975; Kuhar, 1973). In thelast twodecades,extensivestudyof glutamate transport in the brain has defined its ionic requirements, kinetics, and substrate specificity (Hertz, 1979). Glutamate transport is a sodium- and potassium-coupled process that is capable of concentrating intracellular glutamate up to lO,OOO-fold compared with the extracellular environment (Nicholls and Attwell, 1990; Kanner and Schuldiner, 1987). Experiments using tissue homogenates, subcellular fractionations, and cultured cells have all indicated that both neurons and astroglia are capable of high affinity, sodium-

dependent glutamate transport (Hertz, 1979). Pharmacological analysis, using various transport inhibitors, has suggested the existence of several different glutamate transporters (Robinson et al., 1991, 1993b). Recently, three high affinity, sodium-dependent glutamate transporters were cloned: GLT-1 (Pines et al., 1992), EAACI (Kanai and Hediger, 1992), and GLAST (Storck et al., 1992). The cellular localization of these proteins in brain, as inferred from in situ localization of mRNA, was felt to be in glutamatergic neurons for EAACI and in cerebellar Bergmann glia for GLAST. GLT-1 had previously been localized to astroglia (Danbolt et al., 1992; Levy et al., 1993) by immunohistochemistry. In the present study, we have generated anti-peptide antibodies, prepared against the putative intracellular C-terminal domains of each transporter subtype. These have allowed demonstration, by Western blottingand immunocytochemistry,oftheregional,celIular, and subcellular distributions of glutamate transporter subtypes in rat brain with better resolution than possible using in situ hybridization analysis. We demonstrate that each transporter protein is selectively localized to certain populations of neurons or astroglial cells: GLT-1 is specifically localized to astrocytes; EAACI has a neuronal localization that includes nonglutamatergic neurons; and GLAST appears to have a distinct localization to subsets of neurons and astroglia. These findings appear to be consistent with certain anatomical, physiological, and metabolic propertiesof theglutamatergic neurotransmitter system. Results lmmunoblotting Each of the anti-peptide antibodies detected distinct proteins, with antisera to GLT-1 binding a -73 kDa protein, antisera to EAACI detecting a - 69 kDa protein, and antisera to GLAST recognizing a -65 kDa protein in crude homogenates from rat brain (Figure IB). These results were consistent with the molecular masses of proteins predicted from the analysis of cDNAclones.ThemolecularmassesforaIIthreetransporter proteins are slightly higher than the predicted values (Storck et al., 1992; Pines et al., 1992; Kanai and Hediger, 1992). All three antibodies recognized a broad electrophoretic band, which has been seen previously with antibodies to GLT-1 (Danbolt et al., 1992; Levy et al., 1993). Treatment of the homogenates with N-glucosidase F led to a reduction in apparent molecular mass for each of the immunoreactive proteins of about 4-10 kDa (data not shown). These antibodies did not cross-react with proteins of other molecular masses. Anti-GLT-I antibody recognized an apparent doublet, and the high molecular mass species (- 146 kDa) may represent aggregated transporter protein

Nf?URJll

714

A

GLAST EAACl GLT-1

1

B

2

3

1

2

3

1

2

3

205116.5

-

80-

49.5 -

GLAST

EAAC 1

GLT-1

(Danbolt et al., 1992; Levy et al., 1993). A different antibody to GLT-1 that recognizes amino acids 503-519 (H2N-SKSELDTIDSQHRMHED-COOH, provided by Baruch Kanner) produced identical immunoblots in rat brain (data not shown). All immunoreactivity in blots was abolished when antibodies were preabsorbed with their respective synthetic peptides (50 PM), and immunoreactivity in blots was unchanged when antibodies to individual transporters were preabsorbed with synthetic peptides (50 PM) corresponding to C-terminal domains in the other transporter subtypes (Figure 18). The transporter-specific antibodies were used to examine the regional distribution of the EAACI, GLT-1, and GLAST proteins by immunoblotting. There was a differential distribution of both EAACI and GLAST in rat brain (Figure 2), whereas GLT-1 had a more homogeneous brain distribution. The highest levels of EAACI were found in the hippocampus, whereas the lowest levels were in the spinal cord. By contrast, GLAST protein was most abundant in the cerebellum, with lower levels throughout the remaining brain regions and spinal cord. EAACI protein was detected by immunoblot and immunohistochemistry in several nonneuronal peripheral tissues, including small intestine, kidney, and liver (data not shown), in accord with in situ mRNA localization studies (Kanai and Hediger, 1992). GLT-1 and GLAST proteins were not detected in any peripheral tissue under the same conditions.

Figure 1. Anti-Peptide Subtypes

Antibodies

to

Glutamate

Transporter

(A) Amino acid sequences at the C-terminus of each glutamate transporter subtype. Peptides chosen for anti-peptide antibody production are shaded. (6) lmmunoblotting demonstrates the specificity of antibodies toglutamatetransportersubtypes. Rat brain tissue homogenates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies raised against synthetic peptide corresponding to the C-terminus of CLT-1, EAACI, or GLAST. CLAST: GIAST-immunoreactive protein has a molecular mass of -65 kDa (lane 1; 25 ug of protein). lmmunoreactivity is completely abolished when antibodies (0.4 kg/ml) are preabsorbed with 50 u M synthetic GLUT peptide prior to immunoblotting (lane 2). Heterologous peptide (EAACI, 50 PM, or GLT-1 [data not shown]) has no effect on immunoreactivity (lane 3). EAACI: EAACI-immunoreactive protein has a molecular mass of -69 kDa (lane 1; 25 ug of protein). lmmunoreactivity is completely abolished when antibodies (0.6 ug/ml) are preabsorbed with 50 u M synthetic EAACI peptide prior to immunoblotting (lane 2). Heterologous peptide(CLT-I,50 uM) has noeffecton immunoreactivity (lane 3). CLT-1: CLT-I-immunoreactive protein has a molecular mass of - 73 kDa (lane 1; 3 ug of protein). Immunoreactivity is completely abolished when antibodies (0.034 uglml) are preabsorbed with 50 u M synthetic GLT-1 peptide prior to immunoblotting (lane 2). Heterologous peptide (EAACI, 50 uM) has no effect on immunoreactivity (lane 3).

m

Figure 2. Distribution Rat CNS

of Glutamate

SP CORD CBM M IDBRAIN THAI. STRIATUM HIPPO CORTEX Transporter

Subtypes

in the

Aliquots (25 pg of protein per lane for EAACI and GLAST; 3 pg per lane for GLT-I) of tissue homogenates from isolated CNS regions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies to the indicated glutamate transporter subtypes. SP CORD, spinal cord; CBM, cerebellum; THAL, thalamus; HIPPO, hippocampus.

lmmunocytochemistry Overview of the Distributions of C/utamate Transporter Subtypes Sagittal sections (40 urn thick) of whole brains illustrate the distinctive general distributions of glutamate

Localization 715

of Glutamate

Figure 3. Sagittal

Sections

Transporters

of Rat Brain Illustrate

the Regional

Distributions

of EAACI,

GLT-1, and GLUT

Photographic prints were made directly from sections on glass slides. The areas of positive immunostaining appear white. (A) EAACI; (B) GLT-1; (C) GLAST. In the cerebral cortex, laminar patterns of EAACI are present. The hippocampus (HF) is intensely immunoreactive for EAACI and GLT-I. The cerebellar cortex (CC) is enriched in CLAST, GLT-1, and EAACI. Abbreviations: CP, caudate-putamen; DCN, deep cerebellar nuclei; OB, olfactory bulb; SC, superior colliculus. Bar, 2.14 mm.

transporter subtype immunoreactivity(Figures 3A, 3B, and 3C). The precise cellular localizations and ultrastructural patterns demonstrated in semithin (1 urn) and ultrathin plastic sections are unique for each subtype (Figure 4). Controls, including anti-GLAST, -GLT-

1, and -EAACl antibodies preabsorbed against their respective synthetic peptide, normal rabbit IgG at comparable dilutions, and omission of primary antibody, resulted in no immunostaining of any cellular elements (Figures 5D, 5E, and 5F).

Nl3NOIl 716

Figure 4. Ultrastructural

Localization

of Glutamate

Transporters

(A) Within deep cerebellar nuclei, EAACI is found at presynaptic boutons (tl and t2) on unlabeled neurons. An unlabeled bouton (t3) also contacts the same neuron. Bar, 0.95 urn. (B) At higher magnification, EAACI-immunoreactive boutons (tl and t2) form symmetrical axosomatic synapses (arrowheads) with this deep cerebellar neuron. Bar, 0.45 pm. (C)Within thestriatum,an axon terminal (t) forms asymmetrical axodendritic synapses with a dendrite(d2) that is EAACI immunoreactive and a dendritic spine (dl) that is unlabeled. Bar, 0.36 urn. (D) A striatal astrocyte (A) shows intense GLT-1 immunoreactivity. Cross-sectional profiles of astrocytic processes are found within the neuropil (arrowheads). Bar, 1.8 urn. (E) At higher magnification, GLT-l-enriched astrocytic processes are ubiquitous within the neuropil (arrowheads) of the striatum. These astrocytic processes ensheathe dendrites and synaptic complexes. Bar, 0.6 urn. (F) GLAST-immunoreactive glial processes (arrowheads) are found within the striatal neuropil. CLAST-positive astrocytic processes often show transporter enrichment in smaller (more distal) branches (arrowheads), whereas larger segments (more proximal) are less enriched in CIAST (asterisk). Bar, 0.3 pm.

CLT-1 GLT-1 immunoreactivity was generally high throughout all brain regions and the spinal cord, but was largely absent from white matter tracts (Figure 36). At the light microscopic level, immunoreactive processes enveloped neurons in many brain regions, for example, forming discrete sheaths around cerebellar Purkinjecells(Figure5C).Althoughglia-likeprocesses were immunoreactive, astroglial somata were rarely stained. Apparently, similar selective staining of astroglial processes by antibodies raised against purified glial glutamate transporter protein was observed by Danbolt et al. (1992). However, at the ultrastructural level, GLT-I-immunoreactive protein was seen in both

astroglial processes 4E), as also observed

and cell bodies (Figures 4D and previously (Danbolt et al., 1992).

EAACl The distribution of EAACI immunoreactivity was selectively enriched within neurons, and levels of EAACI immunoreactivitywere particularly high in the hippocampus, cerebellum, and basal ganglia (Figure 3A). Punctate immunoreactivity within the neuropil was the predominant pattern of staining, although immunoreactive perikarya and proximal dendrites were also present. Ultrastructural analysis revealed that most neuropil staining corresponded to axons, presynaptic terminals, and dendrites (Figures 4A, 48, and 4C). lmmunoreactive neurons showed diffuse

Localization 717

of Glutamate

Transporters

Figure 5. Glutamate Transporter Subtypes Are Differentially Localized within the Cerebellum EAACI (A), CLT-1 (C), and CL&ST(E) immunoreactivities are each present in the cerebellar cortex. The molecular layer (ML) is enriched in all three transporter subtypes. Purkinjecellsareenriched inEAAC1 immunoreactivity. The Purkinje cell layer (PL) contains perineuronal glial arrays of GLT-1 immunoreactivity and CLAST immunoreactivity. The granule cell layer (CL) has diffuse pericellular CLT-1 and light cellular EAACI immunoreactivity, whereas it is devoid of GLAST immunoreactivity, except for rare Colgi II cells, which are faintly immunopositive. Control sections show the specificity of glutamate transporter immunoreactivity in the cerebellum: incubation in anti-EAACl antibody preabsorbed with 5 PM synthetic EAACI oligopeptide (B); incubation in anti-GLT-1 antibody preabsorbed with 5 BM synthetic GLT-1 oligopeptide (D); incubation in anti-GLUT antibody preabsorbed with 5 PM synthetic GLAST peptide (E). In all controls, immunoreactivity was abolished. Bar, 68 Wm.

GL

and granular cytoplasmic labeling, but showed munoreactivity within their nuclei.

no im-

GLAST GLAST had a still different pattern of immunoreactivity and at the light microscopic level appeared to be present in both neurons and glia. Neuropil immunoreactivity was very strong in the molecular layer of

the cerebellum (Figure 3C) and was almost completely absent from the granule cell layer or deep white matter in the cerebellum. Other regions with moderate neuropil immunoreactivity included the hippocampus (including the dentate gyrus and CAI, CA2, and CA3 regions), superiorcolliculus, and substantiagelatinosa of the spinal cord. Lighter immunoreactivity

Neuron 718

Figure 6. The Neuropil

of Deep Cerebellar

Nuclei

Is lmmunoreactive

for the EAACI

and CLT-1 Glutamate

Transporters

(A) EAACI; (B) CLT-1. (C) Many deep cerebellar neurons are intensely immunoreactive for GLAST. Bar, 73 pm (for A-C). (D) In 1 urn thick plastic sections, deep cerebellar neurons are surrounded by large EAACI-immunoreactive, presynaptic boutons (arrows). Bar, 11 urn. (E) In 1 urn thick plastic sections, counterstained with toluidine blue, deep cerebellar neuronal perikarya demonstrate the typical intracytoplasmic perinuclear distribution of granular GLAST-immunoreactive material (arrow). Ultrastructurally, the GLASTimmunoreactive material in neurons is found in Golgi stacks and associated vesicles, but not in the plasmalemma (not shown). Bar, 11 urn.

was seen in all cortical layers, the hippocampus, Purkinje cells, and spinal cord ventral motor neurons. Neuronal localizationof CLASTimmunoreactivitywas distinct from that of EAACI, with intense immunoreactivity in the deep cerebellar nuclei, superior colliculus, dorsal cochlear nucleus, reticular nuclei, and superior olive. At the light microscopic level, GLAST localization in neurons had a distinctive punctate and often perinuclear distribution. Ultrastructurally, neuronal GLAST was confined to perinuclear locations (Figure 6E). CLAST immunoreactivity within the neuropil was entirely due to localization within astroglial somata and processes (Figure 4F). Within astroglia, CLAST was localized to the membrane, with occasional cytoplasmic particulate immunoreactivity. GLAST protein was selectively localized even within

single astroglial processes that enveloped synaptic complexes (Figure 4F). Interestingly, tanycytes, derived from astrocytes, weredevoid of immunoreactivity for all three transporter proteins. Specific Anatomical Patterns of lmmunoreactivity Cerebellum Glutamate transporter su btypes were differentially localized within the cerebellum (Figures 5A, 5C, and 5E). The neuropil of the molecular layer showed dense immunoreactivityfor all three subtypes, butwas especiallydense for GLAST (Figure 5E). Long, radial immunoreactive processes of Bergmann glia that extend vertically through the molecular layer displayed GLAST and GLT-1 immunoreactivity. By electron microscopy, plasma membranes of astrocytic processes

Localization

of Glutamate

Transporters

719

Figure 7. Patterns of Glutamate Transporter lmmunoreactivity within the Cerebral Cortex and Caudate-Putamen Are Distinct and Specific (A) Most large pyramidal neurons in layer Vare intensely immunoreactive forEAAC1, along with strong EAACl immunoreactivity throughout the cortical neuropil. (B) CLT-1 immunoreactivity is present in a patchy pattern throughout the neuropil. (C) Many small to medium sized nonpyramidal neurons throughout the cortex are immunoreactiveforGL4ST. Numbers indicate cortical layers; wm, white matter. The caudate-putamen is enriched in glutamate transporter immunoreactivity. (D) The majority of the neuropil is strongly immunoreactiveforEAACl,althoughoccasional neuronal somataare also immunoreactive for EAACI. (E) CLT-1 immunoreactivity is present uniformly throughout the structure. (F) Very little GLAST immunoreactivity is present in the caudate-putamen neuropil, and many small neurons (arrowheads) have weak immunoreactivity for GLAST. Bars, 120 urn.

Neuron 720

Figure 8. Coronal Sections of Hippocampus Show Differential Patterns of Glutamate Transporter lmmunoreactivity lmmunoreactivities for EAACl (A), CLT-1 (B), and GLAST (C) are all present within the hippocampus. EAACI and CLT-1 are the most intense, whereas there is considerably less immunoreactivity for GLAST. EAACI immunoreactivity is enriched in CAI-CA3, with less reactivity in thedentate gyrus (DC). GLT-1 has a relative uniform distribution. All sections were processed simultaneously, and photomicrographs were made using identical negative and print exposure times. Bar, 577 pm.

Localization 721

of Glutamate

Transporters

were enriched in GLAST and CLT-1 (Figures 4D, 4E, and 4F). These immunopositive processes enveloped synaptic complexes in the molecular layer. Although Purkinje neurons did not contain GLT-1 or GLAST immunoreactivity, their cell bodies were ensheathed by both GLT-1 and GLAST processes (Figures 5C and 5E). Purkinje cell somata were enriched in EAACI immunoreactivity as well as axons in the subcortical white matter. The granule cell layer was almost completely devoid of immunoreactivity for GLAST, although ultrathin sections revealed occasional immunopositive astroglial processes, and rare Golgi cells in the granular cell layer were also immunopositive. EAACI was present in granule cells. Ultrastructurally, there was no evidence for the localization of EAACI in presynaptic axon terminals within the molecular layer. GLT-1 was associated with granule cell profiles, forming the same ensheathment of neurons seen throughout the brain. Deep cerebellar nuclei (Figure 6) were strongly immunopositive for GLAST (Figure 6C). Although deep cerebellar nuclei did not show EAACI immunoreactivity (Figure 6A), neurons were surrounded by presynaptic boutons immunoreactive for EAACI. The EAACI-immunoreactive boutons formed symmetrical, axosomatic synapses, suggesting that they originated from Purkinje cells (Figure 6D). Deep cerebellar nuclei were also enveloped by astroglial processes immunoreactive for GLT-1 (Figure 6B). Cerebral Cortex Within the cerebral cortex, patterns of immunoreactivity for each of the transporter subtypes were conspicuously different (Figures 7A, 7B, and 7C). EAACI immunoreactivity was confined mainly to large pyramidal neurons localized to layers 111111 and V and in the neuropil throughout the neocortex (Figure 7A). However, other smaller neurons in all cortical layers demonstrated light EAACI immunoreactivity. Intense neuropil staining was also observed in the cingulate gyrus. Many small and medium sized nonpyramidal neurons distributed throughout several laminae exhibited light GLAST immunoreactivity (Figure 7C) within the neuronal perikaryal cytoplasm. Atthe ultrastructural level, in neurons, GLAST immunoreactivity was localized only to the Golgi apparatus. Numerous astroglial processes were GLAST positive. GLT-1 was present throughout the neocortex: in some regions as a uniform neuropil stain; in other regions, neuropil staining was patchy (Figure 78). Similar patchy staining of the GLT-1 transporter in brain has been described (Danbolt et al., 1992) and likely representsvariable distribution of the transporter. St&turn

Anti-GLT-1 antibodies intensely stained astroglial processes within the basal ganglion neuropil, whereas white matter pathways were less enriched in GLT-1 than gray matter (Figure 7E). In the striatum, immunoelectron microscopy showed intensely immunoreactive astrocyte cell bodies (Figure 4D) and revealed that GLT-I-immunoreactive astrocytic processes en-

sheathed virtually all striatal neuron somata and enveloped synaptic complexes (Figure 4E). Expression of GLAST immunoreactivitywas low in basal ganglia (Figure 7F), although GLAST-positive astroglial processes were visualized ultrastructurally (Figure 4F). EAACI immunoreactivity was localized to the majority of medium sized neurons within the striatum (Figure 7D), but large neurons within this region were not immunolabeled. At the ultrastructural level, EAACI immunoreactivity was enriched in postsynaptic elements (dendritic shafts and spines), but not within presynaptic axon terminals that formed synaptic junctions with EAACI-positive postsynaptic elements (Figure 4C). Hippocampus

Within the hippocampus, the patterns of immunoreactivity for all three transporters were striking: subfields CAI, CA2, and CA3 all exhibited intense immunoreactivity for EAACI within the stratum oriens, stratum pyramidal, and stratum radiatum (Figure BA), but thedentate gyrus was much less immunopositive. GLT-1 immunoreactivitywas particularly intense in all areas within the hippocampus, relative to other brain regions (Figure BB). Similarly, GLAST immunoreactivity was also relatively uniform throughout the hippocampus (Figure BC). In ultrathin sections of the hippocampus, both GLT-1 and GLAST were seen within the membranes of astroglial fibers throughout the neuropil. EAACI immunoreactivity was localized to pyramidal neuron cell bodies and postsynaptic dendrites and spines contacted by axon terminals containing round clear vesicles. Spinal

Cord

Within the spinal cord, dorsal and ventral horns displayed differential patterns of glutamate transporter immunoreactivity (Figure 9). Small neurons and the neuropil in Rexed layer 2 were strongly EAACI immunoreactive, whereas motor neurons displayed moderate immunoreactivity (Figure 9A). Similarly, GLAST was also enriched in the neuropil of Rexed layer 2 and in some motor neurons (Figure9C). GLAST immunoreactivity was also seen in the astroglial cells of the white matter. GLT-1 immunoreactivity was enriched in spinal gray matter astroglial fibers and was particularly intense in Rexed layers l-3 (Figure 9B). Discussion These studies describe the specific cellular localization of the high affinity glutamate transporters in the brain. To visualize the distribution of the individual subtypes of the glutamate transporter proteins, antipeptide antibodies were raised against a synthetic peptide corresponding to the C-terminus of each of the cloned glutamate transporters. The characterization of theseantibodies by immunoblot analysisof rat brain membranes shows that they monospecifically react with protein of 65-73 kDa, consistent with the molecular masses predicted by analysis of their cloned cDNAs (Pines et al., 1992; Kanai and Hediger,

NeWOIl 722

Figure 9. EAACl, CLTl, and GLAST lmmunoreactivities calized within the Spinal Cord

Are Lo-

(A) EAACI; (B) CLT-I; (C) GLAST. These cervical sections show that Rexed layers l-3 (arrows) are enriched in all three transporters. The ventral horn contains neuronal populations immunoreactive for both EAACI and CLAST. Bar, 3ClO pm.

Table 1. Localization Neuronal GLT-1 EAACI CLAST + present; a Confined

of Glutamate

Transporter

Perikarya

+ + - absent. to astroglial

processes

within

1992; Storck et al., 1992). The specificity of these antisera was further demonstrated by immunoblotting and immunocytochemical analyses, which revealed that each antiserum could be blocked with its own specific oligopeptide, but not with other transporter oligopeptides. Our immunocytochemical findings (see Table 1 for summary) on the cellular localizations for each of the transporter proteins are consistent, for the most part, with previous Northern blot and in situ hybridization analyses of the distributions of GLT-1, EAACI, and GLAST (Pines et al., 1992; Storck et al., 1992; Kanai and Hediger, 1992). Previous light and electron microscopic studies using affinity-purified polyclonal antisera and monoclonal antibodies prepared against a different oligopeptide sequence showed that GLT-1 was restricted to astroglia (Danbolt et al., 1992; Levy et al., 1993). We confirm that GLT-1 is widely distributed throughout the brain to astroglial cell bodies and processes. Initial reports on GLAST mRNA suggested that it was restricted to Bergmann glia in the cerebellar cortex (Storck et al., 1992). However, the present light microscopic and ultrastructural studies demonstrate that GLAST protein is localized more widely to glial cells in cerebellar molecular and granule cell layers and in fact to some, but not all, astroglia throughout the whole brain. In addition, there appears to be a highly focal subcellular distribution of the protein. Within a single astroglial process, glutamate transporter protein is localized to regions of synaptic contact, as revealed by ultrastructural analysis. Our hypothesis is that astroglia may be able to target the GLAST protein to the plasmalemma of processes that envelop glutamatergic synapses. Such targeting within astroglial cells suggests that it could be a participator in synaptic plasticity. In fact, astroglia have been directly implicated in the synaptic plasticity of the developing visual cortex (Muller, 1992; Muller and Best, 1989). Neurons also contain GLAST; deep cerebellar nuclei, some of which are excitatory or inhibitory (Fagg and Foster, 1983; Chan-Palay, 1982), are GLAST positive.The neuronal localization of GLASToften appears to bedistinctfrom theneuronal localizationof EAACI. For example, in thecortex, GLAST is localized to many small, nonpyramidal neurons in multiple layers, whereas EAACI is localized predominantly to large

Subtypes

Neuropil

Astroglial

Distribution

+’ + +a

+ -

Generalized Hippocampus > Cortex = Striatum > Cerebellum Cerebellum > > Hippocampus > Cortex > Striatum

the neuropil.

+

Localization 723

of Glutamate

Transporters

pyramidal neurons in layers Il/lll and V. However, in certain neuronal populations GLAST, and EAACI may be colocalized to the same cell type. Studies are underway to assess directly whether there is dual localization of these transporters in neurons. The function of GLAST protein in neurons is unclear, as ultrastructural studies demonstrated that, instead of a typical membrane localization, GLAST appears to be restricted to the Golgi apparatus. No pre- or postsynaptic terminals were GLAST positive. It may be that the C-terminal epitope, recognized by the anti-GLUT antibody, is cleaved during protein processing in neurons, but not in astroglia. Thus, GLAST protein may be in neuronal membranes, but not labeled by an anti-C-terminal antibody. Antibodies prepared to other sequences of the GLAST protein will ultimately address this question. Interestingly, at least two astroglial populations were identified by GLAST and GLT-1 immunoreactivities. In some regions it appears that astrocytes may share this protein, e.g., Bergmann glia in the cerebellum. However, in other brain regions, e.g., the cerebral cortex, GLT-1 appeared to be the primary transporter protein in astroglia. Future studies will examine the possible dual localization of these proteins in individual astroglia. It has been suggested previously that EAACI is restricted to glutamatergic neurons, based on in situ hybridization studies showing transcripts located in the granule cell layer of the cerebellum (Kanai and Hediger, 1992; Kanai et al., 1993). However, our immunohistochemistryof the EAACI protein in thecerebellum reveals that the transporter protein is located in the granular cell layer, molecular layer, and Purkinje cells. Thus, the granule cell perikarya in the granule cell layer contain both the mRNA for EAACI and the protein. The transporter protein may be localized to the granule cell processes (parallel fibers), which are in the molecular layer of the cerebellar cortex. However, our ultrastructural studies of EAACI show that EAACI is enriched postsynaptically within Purkinje cells, but not in parallel fiber terminals within the molecular layer. Therefore, EAACI is not confined to glutamatergic neurons, but is also present in nonglutamatergic neurons, including CABAergic cerebellar Purkinje cells. Cortical pyramidal neurons and cerebellar granule cells, as a group, have been considered to be glutamatergic, as defined by glutamate immunocytochemical localization studies, pathway lesion studies examining glutamate transport and/or release, and electrophysiological studies (Fagg and Foster, 1983; Cotman et al., 1987). Our studies demonstrate that many pyramidal neuronsare not immunoreactivefor EAACI and are only lightly immunoreactive for CLAST, and granule cells do not express EAACI presynaptically. In fact, EAACI appears to be localized in the somatodendritic compartment and not within the presynaptic axon terminals of these cells (e.g., parallel fibers and corticostriatal projections). EAACI was not de-

tected ultrastructurally in presynaptic boutons in regions of the brain known to have glutamatergic synapses, such as the neocortex, hippocampus, striatum, or cerebellar molecular layer. Theseobservations suggest that EAACI is not a presynaptic transporter of glutamatergic neurons. Other presynaptic glutamate transporters may exist and have not yet been identified. Since it appears that nonglutamatergic neurons possess glutamate transporter immunoreactivity, it may not be correct to identify glutamatergic neurons through the use of pathway lesion paradigms. The dogma regarding the high affinity glutamate carrier was that it served to transport the neurotransmitter glutamate from the extracellular space and that it was localized to both surrounding astroglia and the presynaptic terminals of glutamatergic neurons (Hertz, 1979; Nicholls and Attwell, 1990). lmmunolocalization with antibodies to the cloned high affinity glutamate transporters suggests that a revision of that hypothesis is necessary. The postsynaptic localization of EAACI and GLAST to GABAergic neurons, i.e., cerebellar Purkinje cells and deep cerebellar nuclei (Chan-Palay, 1982; Krnjevic, 1982), suggests that these transporter proteins may have additional functions. In addition to serving postsynaptically to clear glutamate, both transporters, due to their localization on GABAergic neurons, could serve to transport glutamate intracellularly as a precursor for y-aminobutyric acid synthesis. Because elevated levels of glutamate can be neurotoxic, efficiently maintaining low extracellular concentrations is vital. Several lines of evidence suggest that inefficient glutamate transport leads to the accumulation of excessive neurotransmitter in the synapse, with subsequent neurotoxicity. Experimentally induced, pharmacological blockade of the glutamate transporter causes neuronal death in both acute and chronic models (McBean and Roberts, 1985; Robinson et al., 1991,1993a; Rothstein et al., 1993). Furthermore, defective glutamate transport may play a role in the chronic loss of motor neurons in the neurodegenerative disorder amyotrophic lateral sclerosis (Rothstein et al., 1992) and in the neurotoxicity associated with cerebral ischemia and epilepsy (Choi and Rothman, 1990; Nicholls and Attwell, 1990). Theoligopeptideantibodies described in this report have high specificity for rat glutamate transporter proteins. All three antibodies also have equivalent cellular specificity in human and primate CNS tissue (unpublished data), and three human glutamate transporters with close sequence and peptide homology with EAACI, GLT-1, and GLAST have been cloned (Fairman et al., 1993, Sot. Neurosci., abstract). Thus, these antibodies will serve as excellent tools to study the regulation and dysregulation of these proteins in both experimental models and in disease states.

Experimental

Procedures

Polyclonal Anti-Peptide Antibodies Synthetic peptides corresponding

to the C-terminal

region

of

Neuron 724

the EAACI, GIAST, and CLT-1 proteins (as shown in Figure IA) were synthesized on an Applied Biosystems 430A Peptide Synthesizer (Applied Biosystems, Inc., Foster CityCA).An N-terminal lysine was added to the peptides to facilitate coupling of the peptide to the carrier protein. After synthesis, the peptides were purified using reverse phase high performance liquid chromatography. Each purified peptide was then coupled to thyroglobulin using glutaraldehyde. Antisera were generated against the proteinconjugated synthetic peptides in New Zealand White rabbits (Hazelton, Denver, PA), as previously described (Martin et al., 1992,1993a, 1993b). The crude antisera were affinity purified (Blackstone et al., 1992) on a column prepared by coupling bovine serum albumin-conjugated transporter peptide to AffiGel 15 (Bio-Rad, Rockville Center, NY), prior to their use in immunoblotting or immunocytochemistry. lmmunohistochemistry Tissue was collected from freshly obtained rat brain and spinal cord, dissected, and homogenized with a Brinkman Polytron in icecold phosphate-buffered saline (PBS). In some experiments tissue was homogenized in an ice-cold buffer containing 20 m M Tris-HCl (pH 7.4), 10% (w/v) sucrose, 20 U/ml aprotinin, 20 ).rg/ ml antipain, 20 @g/ml leupeptin, 1 m M EDTA, and 5 m M EGTA. Homogenates were stored frozen at IO mg of protein per ml at -7ooc. The specificity of the antibodies and the regional distribution of the transporter proteins were evaluated by immunoblotting of rat synaptic membranes. Aliquots of synaptic membranes (S-IO ug of protein) were subjected to SDS-polyacrylamide gel electrophoresis (8% polyacrylamide gels) and transferred by electroblotting to polyvinylidene fluoride membranes (Immobilon P, Millipore, Bedford, MA) by electroblotting (30 V, overnight). Blots were blocked (1 hr) in 0.5% nonfat dry milk, 0.1% Tween 20,50 m M Tris-buffered saline (TBS) at room temperature, then incubated (1 hr) with affinity-purified anti-GLT-1, -GLAST, or -EAACl antibody diluted, respectively, to 0.034 ug/ml, 0.4 ug/ ml, or 0.6 uglml in blocking buffer. Blots were then washed, incubated (1 hr) with horseradish peroxidase-conjugated donkey anti-rabbit IgG (I:5000 in blocking buffer), and finally washed with TBS. The immunoreactive proteins were visualized with enhanced chemiluminescence (Amersham, Arlington Heights, IL). lmmunocytochemistry The brains of 250-300 g male and female Sprague-Dawley rats (n = 7) were prepared for both light and electron microscopic evaluation of GLT-1, GLAST, and EAACI. Rats were perfused intra-aortically with 4% paraformaldehyde, 0.1% glutaraldehyde, 15% saturated picric acid, 2% acrolein, or with 4% paraformaldehyde alone, both prepared in phosphate buffer. Brains were removed, blocked, and postfixed (1 hr, 4OC). Brains for light microscopy were cryoprotected (overnight, 4OC) in 20% glycerol, PBS. Brains prepared for electron microscopy were rinsed in cold PBS. Coronal or sagittal sections (48 pm) were cut on a sliding microtome or vibratome and were transferred to cold TBS (pH 7.2). Preliminary studies showed that permeabilization at this stage with 0.4% Triton X-100 for 30 min did not improve appearance on either light or electron microscopy. Subsequent immunocytochemical steps were identical for both groups of sections, except that Triton X-100 was omitted from sections for electron microscopy. Sections were preincubated (1 hr) with 4% normal goat serum diluted in 0.1% Triton X-100, TBS and were then incubated (48 hr, 4°C) in the affinitypurified anti-transporter antibody, at a concentration of 0.2 ggl ml for GLAST, 0.06 fig/ml for EAACI, and 0.17 @ml for GLT-1 in 0.1% Triton X-100,2% normal goat serum, TBS. Control sections were incubated under the following conditions: transporter antibody preabsorbed overnight with an excess (5 JIM) of either synthetic GLAST, CLT-1, or EAACl peptide; primary antisera omitted; or secondaryantibodyomitted. Following primaryantibody incubation, sections were rinsed (30 min) in TBS, incubated (1 hr) with goat anti-rabbit (Cappel) diluted I:200 in TBS with 2% normal goat serum and 0.1% Triton X-100. After rinsing in TBS,

the sections were incubated (1 hr) in rabbit peroxidase-antiperoxidase complex (Sternberger Monoclonals, Baltimore, MD) diluted I:300 in TBS with 2% normal goat serum. After the final incubation, sections were rinsed (30 min) in TBS and developed using a standard diaminobenzidine reaction. After the disclosing reaction, samples (approximately 2 mmz) were taken from vibratome sections, treated (1 hr) with 2% osmium tetroxide, dehydrated, and flat embedded in resin (58% araldite, 48% dodecenyl succinic anhydride, 2% 2,4,6,-tri(dimethylaminomethyl) phenol). Plastic-embedded sections were mounted on an araldite block and cut into semithin (1 urn) and ultrathin (gold to silver interference color) sections for light and electron microscopy, respectively. Ultrathin sections, stained with uranyl acetate and lead citrate, were viewed and photographed with a Phillips CM12 electron microscope.

Acknowledgments This study was funded in part by grants from NIH-NINDS (NS30886and KO8NS01355), the Muscular Dystrophy Association, and theJay Slotkin Fund for Neuromuscular Research. L. M. is the recipient of a Leadership and Excellence in Alzheimer s Disease (LEAD) award (NIA AC 07914). Correspondence should be addressed to 1. D. Rothstein. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

April

18, 1994.

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