Glutamato

The

Journal

of Neuroscience,

March

1995,

15(3):

1835-1853

Differential Expression Rat Brain: Quantitative
Knut P. Lehre, Anatomical Line M. Levy, University Institute,

of Two Glial Glutamate and lmmunocytochemical
Jon Storm-Mathisen, N-031 7 Oslo, Norway

Transporters in the Observations

Ole P. Ottersen, of Oslo, Blindern,

and Niels C. Danbolt

Glutamate, themajor excitatory neurotransmitter in brain, is almost exclusively intracellular due to the action of the glutamate transporters in the plasma membranes. To study the localization and properties of these proteins, we have raised antibodies specifically recognizing parts of the sequences of two cloned rat glutamate transporters, GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992). Onimmunoblots the antibodies against GLT-1 label a broad heterogeneous band with maximum density at around 73 kDa, while the antibody against GLAST labels a similarly broad band at around 66 kDa in the cerebellum and a few kilodaltons lower in other brain regions. GLT-1 is expressed at the highest concentrations in the hippocampus, lateral septum, cerebral cortex, and striatum, while GLAST is preferentiallyexpressed in the molecular layer of the cerebellum. However, both transporters are present throughout the brain, and have roughly parallel distributions in the cerebral hemispheres and brainstem. Preembedding light and electron microscopical immunocytochemistry shows that both GLT-1 and GLAST are restricted to astrocytes, which appear to express both proteins concomitantly, but in differentproportions in different parts of the brain. Nerve terminal labeling was not observed. Both the amino and carboxyl terminals of GLT-1 and GLAST are located intracellularly, indicating an even number of transmembrane segments. Antibodies against a synthetic peptide corresponding to amino acid residues 2-l 1 of the proposed sequence of GLT-1 recognize the native rat brain GLT-1 protein, confirming thatthe translation initiation site is at the first ATG. [Key words: cotransport, neurotransmitter transport, astrocytes, glutamate, anti-peptide antibodies, immunocytochemistry]

The resting extracellular concentration of the neurotransmitter (Q-glutamate has to be kept low, not only to allow for activation of glutamate receptors (Schoepp and Conn, 1993; Seeburg, 1993) to fluctuate with the releaseof glutamate from the nerve
Received Mar. 28, 1994; revised Aug. 12, 1994; accepted Aug. 22, 1994.

terminals, but also because excessive activation of glutamate receptors can lead to neuronal damage (Choi, 1992). The low extracellular concentration is due to the action of “high affinity” sodium dependent glutamate transporters (Balcar and Johnston, 1972; Logan and Snyder, 1972; Johnston, 1981;Kanner and Schuldiner, 1987; Nicholls and Attwell, 1990) located in the plasma membranes of both neurons (Divac et al., 1977; StormMathisen and Iversen, 1979; Taxt and Storm-Mathisen, 1984; Garthwaite and Garthwaite, 1988; Gundersen et al., 1993) and glial cells (Schousboe, 1981; Wilkin et al., 1982; Danbolt et al., 1992; Levy et al., 1993a). Several glutamate uptake systems have been described(for review, see Danbolt, 1994). Three different eukaryotic glutamate transporters constituting a “new” protein family were cloned in 1992-GLAST (Storck et al., 1992), GLT-1 (Pines et al., 1992), and EAAC- 1 (Kanai and Hediger, 1992)-but the localizations and relative distributions of the proteins have not been worked out. Although the antibody used to clone GLT has previously been used forimmunocytochemistry (Danbolt et al., 1992), the possibility existed that this could detect also other members of the family. The proteins have about 55% amino acid identity with each other and 34-39% identity with ASCT- 1, a human sodium dependent neutral amino acid transporter (Arriza et al., 1993; Shafqat et al., 1993). Originally, different numbers of transmembrane ol-helices were predicted for...