Functions of the two glutamate transporters GLAST and GLT-1 in the retina

Abstract

In the retina, the glutamate transporter GLAST is expressed in Müller cells, whereas the glutamate transporter GLT-1 is found only in cones and various types of bipolar cells. To investigate the functional role of this differential distribution of glutamate transporters, we have analyzed GLAST and GLT-1 mutant mice. In GLAST-deficient mice, the electroretinogram b-wave and oscillatory potentials are reduced and retinal damage after ischemia is exacerbated, whereas GLT-1-deficient mice show almost normal electroretinograms and mild increased retinal damage after ischemia. These results demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and that both GLAST and GLT-1 play a neuroprotective role during ischemia in the retina.L-Glutamate is the major excitatory neurotransmitter in the mammalian retina (1). High-affinity glutamate transporters are believed to be essential for terminating synaptic transmission as well as for keeping the extracellular glutamate concentration below neurotoxic levels (1, 2). Five subtypes of glutamate transporter (GLAST, GLT-1, EAAC1, EAAT4, and EAAT5) (3-8) have been cloned, but the contributions of individual transporter subtypes to retinal function are poorly understood. Studies have been hampered by the lack of subtype-selective glutamate transporter drugs. As an alternative approach, we have analyzed GLAST-and GLT-1-deficient mice (9, 10). Our results demonstrate that GLAST is required in retinal signal transmission at the level of the photoreceptor and bipolar cell and that GLAST and GLT-1 are crucial for the protection of retinal cells from glutamate neurotoxicity. MATERIALS AND METHODSImmunohistochemistry. Mice were anesthetized with diethyl ether and perfused transcardially with saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer containing 0.5% picric acid at room temperature. Eyes were removed and postfixed overnight in the same fixative, and 7-m-thick paraffin or frozen sections were cut and mounted onto gelatin-and poly-L[D]-lysine-coated slides. The sections were incubated overnight with an affinity-purified rabbit polyclonal antibody against the carboxyl-terminal sequence of the mouse GLAST (1.0 g͞ml) (KKPYQLIAQDNEPEKPVAD-SETKM) (11, 12), an affinity-purified rabbit polyclonal antibody against the rat GLT-1 (0.2 g͞ml) [anti-B12; gift from N. C. Danbolt] (13), or a mouse monoclonal antibody against glutamate synthetase (GS) (2.0 g͞ml) (Chemicon) at room temperature. The sections were then incubated with biotinylated goat anti-rabbit IgG (Nichirei, Tokyo) for GLAST and GLT-1 or biotinylated rabbit anti-mouse IgG (Nichirei) for GS for 1 hr, followed by further incubation with streptavidin-Texas red (NEN) for 30 min at room temperature. Sections were examined by a confocal laser scanning microscope (Molecular Dynamics).Electroretinograms (ERGs). Mice (9-11 weeks old) were anesthetized by intraperitoneal injection of a mixture of xylazine (10 mg͞kg) and ketamine (25 mg͞kg). The pupils were dilat...

Glutamate uptake in retinal glial cells during diabetes

“…It is well known that GLAST provides the bulk of glutamate uptake within the retina [23,24,38]. Indeed, loss of function of GLAST results in a major loss of retinal structure and function.…”

“…At the molecular level, five glutamate transporters have been cloned: excitatory amino acid transporters (EAAT) 1-5, all of which have been identified in the retina [13,14,[18][19][20][21][22]. Although the function of specific transporter types is contentious, it is clear that GLAST (EAAT1) is the predominant transporter for removal of glutamate within the retina and defects in its function lead to major deleterious effects on vision [20,23,24].…”

The upregulation of GLAST-1 is an indirect antiapoptotic mechanism of GDNF and neurturin in the adult CNS

“…17 has been localized to cone photoreceptors and cone bipolar cells in the mammalian retina. 18 GLT-1-deficient mice show increased neuronal damage after retinal ischemia, 19 suggesting that GLT-1 plays an important role in CNS neuronal apoptosis.…”

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

“…The GLAST Ϫ/Ϫ and GLT1 Ϫ/Ϫ mice have been particularly useful in analyzing the role of glial glutamate transporters in excitotoxicity (Harada et al, 1998;Mitani and Tanaka, 2003). Because GLAST is a major glutamate transporter in the mammalian retina, loss of GLAST is expected to render the retina highly susceptible to excitotoxic damage.…”

“…Because GLAST is a major glutamate transporter in the mammalian retina, loss of GLAST is expected to render the retina highly susceptible to excitotoxic damage. However, the GLAST Ϫ/Ϫ mouse retina shows a benign phenotype, although it is more sensitive to prolonged ischemia than the normal retina (Harada et al, 1998). The reason for the lack of excitotoxic damage is unknown.…”