Colocalization of GLUT3 and Choline Acetyltransferase Immunoreactivity in the Rat Retina

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Biochemical and Biophysical Research Communications 256, 505–511 (1999)

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olocalization of GLUT3 and Choline Acetyltransferasemmunoreactivity in the Rat Retina

akashi Watanabe,*,†,1 Shinya Nagamatsu,‡ Satsuki Matsushima,*akaaki Kirino,†,§ and Hidemasa Uchimura*

Department of Clinical Pathology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; †CRESTCore Research for Evolutional Science and Technology), Japan Science and Technology Corp., Kawaguchi 332-0012,apan; ‡Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; andDepartment Neurosurgery, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

eceived February 8, 1999

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Toward elucidating the functional aspects ofLUT3, a primary neuronal glucose transporter iso-

orm in the vertebrate central nervous system, thistudy examined its expression in cholinergic amacrineells made identifiable by the presence of acetylcho-ine-synthesizing enzyme, choline acetyltransferaseChAT), in the rat retina. Double-immunofluorescencetaining of adult rat retinal tissue with anti-GLUT3nd anti-ChAT antibodies revealed characteristictratified GLUT3 immunoreactivity (GLUT3-IR) in thenner plexiform layer (IPL) that was identical to therborization pattern of ChAT-positive neuronal pro-esses there. In addition, approximately 30–50% of in-ensely GLUT3-immunoreactive cell bodies in the in-er nuclear layer and ganglion cell layer showedhAT-IR, while the majority of ChAT-positive cell bod-

es were also intensely GLUT3 immunoreactive. Anal-sis at the cellular level using retinal cells in cultureevealed similar findings. These results collectivelyndicate that cholinergic amacrine cells constitute the

ajor component of GLUT3-expressing cells in the ratetina. It is expected that the link demonstrated hereetween GLUT3 expression and cholinergic amacrineell population will provide clues for further analyz-ng GLUT3 function in the retina. © 1999 Academic Press

Key Words: GLUT3; choline acetyltransferase; retina;holinergic amacrine cells.

As the mammalian retina requires a substantial en-rgy supply to maintain its unique neuronal activity,nd glucose is the primary substrate for fueling metab-lism in this organ [1], this indicates that glucose ho-eostasis is essential for the retinal function [2–4].1 To whom correspondence should be addressed at Department of

linical Pathology, Kyorin University School of Medicine, 6-20-2hinkawa, Mitaka, Tokyo 181-8611, Japan. Fax: 181-422-79-3471.-mail: [email protected].

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alian cells primarily occurs via a saturable, stereo-pecific, facilitated-diffusion process mediated by aamily of integral membrane proteins referred to asacilitative glucose transporters (GLUTs) [5–7]. Ithould therefore not be a surprise that GLUTs playmportant roles in the regulatory mechanisms thatnsure sustained and stable glucose supply to the ret-nal tissue, although their precise functions as well as

olecular mechanisms regulating their expression re-ain largely unclear. Several GLUT isoforms have

hus far been identified, each demonstrating distincthysiological properties and tissue distribution [5–7].f these, GLUT1/2/3 have been shown to be present in

he rat retina [8–10]. GLUT1, mainly localized on pig-ent epithelial cells and retinal vascular endothelial

ells, is known to be involved in glucose transportcross the blood-retina barrier [8, 11], while GLUT2,resent at the apical ends of Muller cells, appears tolay role(s) in anterior and/or posterior transport oflucose within the retina [9].GLUT3, mainly present on neuronal cells [12–14],

as been considered to be a primary neuronal GLUT inhe mammalian central nervous system [5–7]. How-ver, its normal function(s) along with its significancender various pathological conditions associated withltered availability of glucose remain largely unknown.n the rat retina, GLUT3 is detected mainly in theeuronal processes located in the inner and outer plex-

form layers (IPL and OPL), while some cell bodiesithin the inner nuclear layer (INL) also express it

10]. In our previous study, we observed the presence ofwo prominent, narrowly stratified immunoreactiveands in the IPL as well as diffuse staining there [10].uch an expression profile of GLUT3 in the retina isarticularly noteworthy because neuronal processes ofarious functional subsets of retinal neurons arenown to demonstrate characteristic laminar arboriza-

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

tion patterns within the INL [15]. With this in mind,wtsfGt

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Vol. 256, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

e surmised that comparative evaluation of arboriza-ion patterns of various retinal cell subsets with thoseeen in GLUT3 immunolabelling would provide cluesor elucidating particular retinal cell types expressingLUT3, and accordingly, allow us to better understand

he function of this unique GLUT isoform.Here, we focus our attention on the cholinergic am-

crine cell population made identifiable by the pres-nce of the acetylcholine synthesizing enzyme, cholinecetyltransferase (ChAT), since the neuronal processesf this cell type have been reported to show a stratifiedrborization pattern highly similar to that seen inLUT3 immunolabelling [15–17]. That is, toward elu-

idating functional aspects of GLUT3 in the retina, aouble-immunofluorescence method using anti-GLUT3nd anti-ChAT antibodies is employed to examinehether GLUT3 is expressed by cholinergic retinal

ells.

ATERIALS AND METHODS

Animals. Timed-pregnant and adult (200–250 g) Sprague–awley (SD) rats were obtained from Clea Japan (Tokyo, Japan). To

ime the pregnancy, female rats were caged with males overnightnd then removed, with this being taken as day 0 of pregnancy.nimals were maintained on a 12/12-h light/dark cycle in a

emperature- and humidity-controlled room with access to food andater ad libitum. All animal experiments were performed in accor-ance with the Association for Research in Vision and Ophthalmol-gy (ARVO) statement for the use of animals in ophthalmic andision research.

Antibodies. To identify GLUT3, we used rabbit anti-rat GLUT3ntiserum (ALM3-C) raised against the C-terminus oligopeptide pre-icted from the DNA sequence of rat GLUT3 [18, 19]. ALM3-Cecognizes GLUT3 in the rat retina as a protein with an averageolecular weight of approximately 44 kDa in immunoblot analysis

18]. Cholinergic amacrine cells were identified using a monoclonalntibody directed against ChAT (1E6, Chemicon). ChAT is known toe a specific marker for this cell type in the rat retina as well as inhe retina of many other vertebrates [16, 17, 20].

Retinal cell culture. Pregnant SD rats at 18 days of gestationere sacrificed by cervical dislocation under deep anesthesia withiethyl ether, and the embryos were removed. Neural retinae werehen dissected using a dissecting microscope and incubated for 10in at 37°C in Ca21- and Mg21-free Hank’s balanced salt solution

HBSS; Gibco BRL) containing 0.125% (w/v) trypsin (Sigma). Theissue was transferred to Ca21- and Mg21-free HBSS containingNase (0.04 mg ml21; Sigma), soybean trypsin inhibitor (0.25 mgl21; Sigma), and bovine serum albumin (3 mg ml21; Sigma), and

hen dissociated into a single cell suspension by gentle triturationhrough a Pasteur pipette. After washing the cells with minimumagle’s essential medium (Gibco BRL) containing 10% fetal calferum (FCS; Gibco BRL), 200,000 cells were suspended in 40 ml ofulbecco’s modified Eagle’s medium (Gibco BRL) supplemented with0% FCS and plated on poly-D-lysine- and laminin-coated glassoverslips (Collaborative Biomedical) in 24-well tissue culture platesFalcon). After incubating for 60 min at 37°C to allow the cells todhere, 500 ml of the same medium was added and the cells wereultured for 18 d in a humidified atmosphere of 5% CO2 and 95% airt 37°C. The day after dissociation, 500 ml of the same medium wasdded to each well. The culture was maintained by replacing half theolume of culture medium with fresh solution every other day.

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ubjected to kainic acid (KA) treatment and five served as controls.ntraocular injection was performed on animals anesthetized byntraperitoneal administration of 5 mg of pentobarbital sodiumAbott) in 1 ml of saline. KA (Sigma) was dissolved in phosphate-uffered saline (PBS) at a concentration of 10 mmol/l and the solu-ion was adjusted to pH 7.2. Using a heat-pulled glass capillaryonnected to a microdispenser (Drummond Scientific Co., Broomall,A), the prepared KA solution (5 ml) was injected into the vitreoushamber of one eye over a period of 1–2 min. PBS (5 ml) was similarlynjected into one of the eyes of the controls. Rats were sacrificed

d later and their eyes processed for double-immunofluorescencetaining.

Immunofluorescence staining. Adult SD rats injected with KA orBS were perfused transcardially with 300 ml of PBS followed by theame volume of ice-cold fixative containing 4% formaldehyde in 0.1

phosphate buffer (PB, pH 7.4) under deep anesthesia with diethylther. Eyes were enucleated immediately, with posterior eye cupseing dissected and immersion-fixed in the same fixative for 1 h at°C. Samples were cryoprotected with a graded concentration ofucrose in PBS at 4°C, embedded in OCT compound (Miles), androzen in liquid N2. Frozen sections (12 mm) were cut on a Milesryostat, transferred onto poly-L-lysine coated slides, and air driedor 30 min at room temperature (r.t.). After washing in PBS for 10in, sections were treated with 10% normal sheep serum in PBS for

0 min at r.t. and then with anti-ChAT monoclonal antibody (Chemi-on, diluted 1:150) overnight at 4°C, followed by incubation withheep anti-mouse immunoglobulin coupled to fluorescein (Sigma,iluted 1:40) for 1 h at r.t. Anti-ChAT and fluorescein-conjugatednti-mouse immunoglobulin antibodies were both diluted in PBSontaining 4% FCS and 0.1% sodium azide. Double-immunolabellingas performed by further incubation with 10% normal goat serum

or 10 min at r.t., followed by incubation with ALM3-C (anti-GLUT3)ntibody (diluted 1:200) for 3 h and then with anti-rabbit immuno-lobulin coupled to Texas-red (Molecular Probe, diluted 1:400) forh, both at r.t. ALM3-C and Texas-red-conjugated anti-rabbit im-unoglobulin were diluted in PBS containing 4% FCS, 0.1% Triton-100, and 0.1% sodium azide. Control stainings were similarlyarried out, without adding the primary antibodies to dilution buff-rs. Stained sections were mounted with glycerol containing 0.1%araphenylenediamine to prevent bleaching, examined under a Zeissxioplan fluorescence microscope with Nomarski optics, and photo-raphed with T-max film (400ASA, Kodak).To perform double-immunofluorescence staining of retinal cells

ultured on glass cover slips, the cells were fixed in 4% formaldehyden 0.1 M PB (pH 7.4) for 10 min at r.t. and stained. The stainingrocedure was similar to that used to stain frozen sections, excepthat cells were incubated with primary antibodies for 2 h and withecondary antibodies for 40 min, both at r.t.

ESULTS

Colocalization of GLUT3 and ChAT in vivo. To ex-mine whether GLUT3 colocalizes with ChAT in theat retina, frozen sections of formaldehyde-fixed adultD rat posterior eye cup were subjected to double-

mmunofluorescence staining with ALM3-C (anti-LUT3) antibody and then anti-ChAT antibody. Asreviously reported [10], GLUT3 immunoreactivityGLUT3-IR) is mainly localized in the IPL and OPL ofhe retina (Figs. 1D and 1E). That is, diffuse stainingppears in the IPL and OPL along with two prominent,arrowly stratified intensely GLUT3-immunoreactiveands; one being situated close to the center of the IPLnd the other between this band and the posterior

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FIG. 1. Double-immunofluorescence staining of SD rat retina with ALM3-C (anti-GLUT3) and anti-choline acetyltransferase (ChAT)ntibodies. Vertical cryostat sections of formaldehyde-fixed, posterior eye cups of adult rats were immunolabeled with anti-ChAT antibodynd then ALM3-C antibody (D, E, F). Control staining (A, B, C) was carried out using dilution buffer alone. Stained sections werehotographed using Nomarski (A, D), Texas red (B, E), or fluorescein (C, F) optics. A–C and D–F, respectively, show identical fields. A, B,: Control stainings in the absence of the primary antibody revealed no significant immunoreactivity for either GLUT3 or ChAT. D, E, F: Twoarrowly stratified immunoreactive bands appearing in the inner plexiform layer (IPL) showing identical staining patterns for GLUT3 andhAT immunolabelling (E, F, arrows). Cell bodies showing immunoreactivity for both GLUT3 and ChAT are present in the inner nuclear

ayer (INL) and ganglion cell layer (GCL) (E, F, arrowheads). An intensely GLUT3-immunoreactive cell which is not ChAT positive is shownE, F, white arrowheads). In cell bodies, GLUT3-IR is mainly localized on cell surfaces, while ChAT-IR is mainly present within theytoplasm. ONL, outer nuclear layer; OPL, outer plexiform layer; PE, pigment epithelium. Bar 5 50 mm.

boundary of the IPL (Figs. 1D and 1E). GLUT3-IR wasall(aisa

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lso detected in many cell bodies in the inner nuclearayer (INL) and in some cell bodies in the ganglion cellayer (GCL), while cell bodies in the outer nuclear layerONL) did not exhibit such immunoreactivity (Figs. 1Dnd 1E). Although the staining level in most cell bodiesn the INL and GCL was very faint, a small proportionhowed relatively intense GLUT3 staining (Figs. 1Dnd 1E).ChAT immunostaining of the same section showed

wo narrowly stratified immunoreactive bands appear-ng in the IPL as has been reported [15–17, 20], al-hough neither the IPL nor OPL exhibited diffusetaining (Figs. 1D and 1F). Comparative evaluationevealed that both these bands were identical to theLUT3-IR bands in terms of their localization within

he INL and staining pattern (Figs. 1E and 1F). Inddition, two distinct populations of ChAT-expressingells were observed, i.e., those with cell bodies localizedn the innermost layer of the INL and those with cellodies in the GCL (Figs. 1D and 1F). Of particularnterest, the majority (approximately 80–90%) ofhAT-positive cell bodies in the INL and GCL corre-ponded to the intensely GLUT3-positive cells (Figs.E and 1F), while the proportion of ChAT-immuno-eactive cells among intensely GLUT3-positive cellsas approximately 30–50%. It is also noteworthy thatdistinct GLUT3 and ChAT localization pattern was

oted in cell bodies; i.e., GLUT3-IR was mainly local-zed on cell surfaces, while ChAT-IR was mainlyresent within the cytoplasm (Figs. 1E and 1F). Con-rol stainings in the absence of the primary antibodyevealed no significant immunoreactivity for eitherLUT3 or ChAT (Figs. 1A, 1B, and 1C).

Colocalization of GLUT3 and ChAT in cultured ret-nal cells. To further examine colocalization ofLUT3 and ChAT at the cellular level, dissociatedmbryonic day 18 (E18) retinal cells were cultured initro for 18 days and analyzed for the presence ofLUT3 and ChAT using double-immunofluorescence

abeling with anti-ChAT and then anti-GLUT3 anti-odies. Results show that some cultured retinal cellsxhibit ChAT-IR in their cell bodies and neuronal pro-esses, although the expression level in the latter isess intense (Figs. 2A and 2C). While the double-abeling analysis indicates that neuronal processes andell bodies of ChAT positive cells are also essentiallyLUT3 immunoreactive, GLUT3-expressing cell bod-

es as well as neuronal processes with no detectablehAT-IR were also found (Figs. 2B and 2C).

GLUT3 expression in kainic acid-treated rat retina.ince intraocular injection of KA is known to produce aignificant loss of cholinergic amacrine cells in the ret-na [21, 22], we examined the expression of GLUT3 inA-treated SD rat retina to determine whether deple-

ion of cholinergic retinal neurons has any effect on

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A into one of the eyes of adult SD rats, posteriorye cups were dissected and subjected to double-mmunofluorescence labeling with anti-GLUT3 andnti-ChAT antibodies. As the control, PBS was injectednto one of the eyes of adult SD rat, which were pro-essed similarly. Comparative analysis showed that (i)A-treated retinae were remarkably reduced in thick-ess (Fig. 3D vs Fig. 3A), (ii) the reduced thickness wasainly due to thinning of the IPL and INL associatedith a reduction in the number of cells in these layers

Figs. 3A and 3D), and (iii) cell number in the GCL wasignificantly lower in KA-treated retinae than that inontrols (Figs. 3A and 3D). In comparison with normalon-injected retinae, GLUT3 and ChAT double label-

ng in controls showed no significant difference in ei-her GLUT3-IR or ChAT-IR (Figs. 1D, 1E, and 1F andigs. 3A, 3B, and 3C). Regarding ChAT-IR in treatedetinae, ChAT-IR both in cell bodies and neuronal pro-esses disappeared completely (Fig. 3E). GLUT3-mmunolabelling on the same section revealed that KAreatment also produced a significant decrease inLUT3-IR, i.e., only trace GLUT3-IR was present in

he IPL and in limited areas of the GCL (Fig. 3F).

ISCUSSION

Results of our double-immunofluorescence labelingnalysis demonstrated that the two characteristicLUT3-immunoreactive strata appearing in the IPL,eing the major component of GLUT3-IR in the ratetina [10], were precisely identical to the two narrowlytratified ChAT-immunoreactive strata in the IPL.uch correlation appears to indicate that at least someLUT3-positive cells are cholinergic; and accordingly,LUT3- and ChAT-IR could colocalize on some of the

ell processes which arborize within two narrow planesn the IPL. Another possibility is that the identicaland profiles are the result of neuronal processes ofeparate retinal cell populations expressing GLUT3 orhAT respectively stratifying within the same planes

n the IPL. In fact, ON and ON-OFF direction-selectiveanglion cells, for example, are known to costratifyith cholinergic amacrine cells within the IPL [23]. Weelieve, however, that the former possibility is moreikely since GLUT3- and ChAT-IR are apparently co-ocalized in some cell bodies located in the INL andCL. Consistent with previous reports [17,24], two

ubclasses of ChAT-positive cells were identified: oneaving their cell bodies in the innermost layer of theNL and the other with their cell bodies in the GCL,orresponding to the OFF- and ON-type cholinergicmacrine cells, respectively. The majority of cholin-rgic amacrine cells in the INL and GCL were in-ensely GLUT3 positive, whereas approximately 30–0% of intensely GLUT3-positive cells in the INL andCL were ChAT positive. Similar findings were ob-

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ained by double-labeling analysis of cultured retinalells, which demonstrates that the majority of ChAT-ositive cells were also GLUT3 positive. In this casegain, some GLUT3-positive cells showed no signifi-ant ChAT-IR. These in vivo and in vitro findingslearly indicate that the majority of cholinergic retinalells express GLUT3, and that these GLUT3-positiveholinergic cells constitute a major subpopulation ofLUT3-expressing cells. In an animal model whereany INL cells including ChAT-immunoreactive cellsere depleted by intraocular injection of KA [21, 22],

he two ChAT-immunoreactive bands in the IPL com-

FIG. 2. Double-immunofluorescence staining of cultured retinal c8 SD rat retinal cells were cultured in vitro for 18 days, immunhotographed using Nomarski (A), Texas red (B), or fluorescein (C) opetinal cells exhibiting ChAT-IR in their cell bodies (C, arrowheads)odies (B, C, arrowheads) as well as neuronal processes (B, C, arroresent (A, B, C, white arrowheads). Bar 5 50 mm.

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letely disappeared and GLUT3-IR was attenuated totrace presence. Such results are consistent with the

iew that cholinergic amacrine cells constitute the ma-or cellular component of GLUT3-expressing cells inhe rat retina. These results are noteworthy becausehis is the first evidence demonstrating that GLUT3xpression is related to a particular functional subsetf neurons in the mammalian central nervous sys-em. Presently, however, the characteristics of otherLUT3-positive cells remain unknown. The diffuseLUT3 staining appearing throughout the IPL, could,owever, reflect its expression in some subsets of dif-

with ALM3-C and anti-ChAT antibodies. Dissociated embryonic dayained with anti-ChAT antibody and then ALM3-C antibody, ands. A–C show identical fields. The majority of ChAT-positive cultured

neuronal processes (C, arrows) also expressed GLUT3 in their cell. GLUT3-positive cells that are not ChAT-immunoreactive are also

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use amacrine cells [25] which are distinct from strat-fied amacrine cells including cholinergic amacrineells in that they extend their processes diffuselyhroughout the IPL.

Although GLUT3 is predominantly expressed in ro-ent brain [13], its regional distribution within theNS appears to be rather heterogeneous [14, 19, 26,7]. For example, GLUT3 expression is absent in theateral olfactory tract of the rat, whereas in the neigh-oring pyriform cortex, strong expression has been ob-erved [19]. Since the level of GLUT3 mRNA or proteinxpression generally reflects the glucose utilizationate as determined by the 2-deoxyglucose technique,uch regional heterogeneity in GLUT3 expression in

FIG. 3. Double-immunofluorescence staining of kainic acid (KA)ays after the injection of KA into one of the eyes of adult SD rats, thetaining (D–F). As controls, one of the eyes of other adult SD rats wasmmunolabeled with anti-ChAT antibody and then ALM3-C antibuorescein (C, F) optics. In comparison with controls, KA treatmenthickness of the IPL and INL (A, D). Although KA-treated retinaeeuronal processes (D, E), the control retinae (A, B) showed no signF). KA treatment also caused a significant decrease in GLUT3-IR,

510

he brain has been attributed mainly to variable glu-ose utilization, or in other words, variable metabolicates in different brain regions [19, 26]. Accordingly,uch observations may suggest that cholinergic ama-rine cells are among the most metabolically active ofhe various subsets of retinal neurons. Consistent withhis view, when the retina is exposed to flashing light,ts inner part is known to show a considerable increasen glucose requirement [28, 29]. Since exposure toashing light simultaneously increases ACh releaserom the retina [30], the increase in glucose require-

ent in this region might reflect the activities of met-bolically active cholinergic amacrine cells includingN- and/or OFF-types. And, if cholinergic amacrine

ated SD rat retina with ALM3-C and anti-ChAT antibodies. Sevensterior eye cup was dissected and processed for immunofluorescenceected with PBS and similarly processed (A–C). Frozen sections were

and photographed using Nomarski (A, D), Texas red (B, E), oratly reduced the retinal thickness mainly due to a reduction in theowed complete disappearance of ChAT-IR in both cell bodies andnt changes compared to normal non-injected retinae (Figs. 1D andh only traces being present in the IPL and GCL (F). Bar 5 50 mm.

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nce of GLUT3 in these cells would be essential fornsuring a stable and sustained glucose supply. Oxy-en profile analyses of the retina have shown that theNL and its neighboring regions where GLUT3 mainlyocalizes could be the balance point between the two

ajor retinal vascular systems [31]—the retinal andhoroidal vasculatures—which suggests that glucoseoncentration in this region might be lower relative tohe rest of the retina such that cells in this regionould necessarily be most vulnerable to insults such as

schemia or hypoglycemia where glucose availabilityecomes limited. If so, then the presence of GLUT3ould be most beneficial in these cells since it has been

eported to operate more efficiently under low glucoseoncentrations, i.e., GLUT3 shows a low Km value forlucose [32, 33].In conclusion, based on our finding that rat retinal

holinergic amacrine cells constitute the major compo-ent of GLUT3-expressing cells, we propose that theunctional significance of GLUT3 expression on cholin-rgic amacrine cells is most likely related to the met-bolic rate of this cell type as has been postulated forLUT3 in the brain. As such, the link demonstratedere between GLUT3 expression and the cholinergicetinal cell population may in fact provide necessarylues to fully explain the function of GLUT3, while alsoroviding a better understanding of the true nature oflucose homeostasis within the retina.

CKNOWLEDGMENTS

Sincere gratitude is extended to Dr. Anthony L. McCall for hisenerous gift of ALM3-C antibody. This work was supported in party a grant for “Research for the Future Program #97I00401” from theapan Society for the Promotion of Science.

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