Exp. Eye RCJS.(1978) 27, 435-44.4

Glutamine Synthetase in the Developing Rat Retina: An Immunohistochemical Study

Thr distribution of glutamine xynthetase ((3) was stlltlietl in rat retinas from the clay 01 birth to adulthood by means of immnnohistot~hemistr~-. (3 was present in the pigment cpithelium in the newborn rat, and over the next few days it was demon&at4 around blood wssels and within small glial cells. The enzyme n-as first detected in perikarya and processes of Alfiller cells on day 5. The adult C:S pattern was acyuirtd by day 12. except for persistence of( 3 in the pigment epithelium. (3 in the pigment epith&um gradually diminished to trace amounts between day 12 and 2 months. Additionally. (:S was found in the inner cpitheliurn of the ciliary body and posterior epithelium of the iris during t’he developmental period and in adults. Our result,s indicate that, the dramatic rise in retinal C% biochemically tlemonstrated hy other workers occurs exclusively in RIiilltrr cells. Moreover. the complete actl”isit,ion of GS in rat, ret,ina corresponds chronologically with certain mat’urational evc,nts including the onset ofthe electroretinographic reslmnse. ‘The role of ($8 in the pigment eptttlelium and extraretinal structures is uncertain hut it may denote their involvement in m,lc,opol?;ssrrharide metabolism. lir!/ words: glut,amine synthet.ase; developing rrtina : iml,llrnohist.ocllenlist.r~: Jliiller ~41.

1. Introduction Gltttatttine synthetase (GS) has been the suljject ot’ a nuntlm of developtnental stuclies in retina. B striking increase in the activity of this enzynte has l)een dentonstrstcd in the rat and chick retina concurrent with maturation (Rudnick and Waelsch. 1955: Moscona and Hubby, 1963; C’hader. 1971). Mweover, hormonal induction of GS in the developing retina has provided a mluahlr tttotlel for t,he study of mechanisms controlling gene expression (Moscona, 1973). dtlditionall~+, GR in brain (Van den Berg. 1970) and presuntably in retina is involved in t.he tnetaholistn of the putative neurotransittitt,ers y-atninohut,yrie acid (G,4B-A) and glutan~ate (Starr, 1974 : Kentttdyy Neal and Lolley. 197i). Similarly. C;S is critical in t’he metabolism of anltllonia (Van den Berg, 1970; C’hader. 197 1). ant1 provides a source of glut~atnint~ necessary for the retinal synthesis of tttucol)ol~sacch;trides (Mazlen, Mullenberg and O’Brien, 19iO ; Smith and Newotne. 1958). In view of the importance of GS in retinal metabolism and its role in our ttnderstancling of enzyme induction, a preciseanatomical topography of this enzyme during tlevrlopent is desirable. In this report we descril,e t,he results of an itntnunohist~ochetllical stucly of GS in rat, retina front the newl~trn through 2 months of age. 2. Materials and Methods of 0014

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3. Results The fixation protocol resulted in cxcelltmt t,issue ])reservatjitm as j~~tlgetl lty histological criteria and lw the capeit\of the tissue to withstand t’he iJlllllunohistt)chemical procedures. ‘The histological features of retinal clevelol~met~tol~servc~l II> light microscope were essentially the mue as those tlescrihetl IJy Wlipidnian ;tJltl Kuwabara (19Sb). Until day 4. an inner ganglion cell layer was selmatetl frOrlJ it layer of undifferentiated neuroblastic cells tby a narrow inner plexiform layer. The innermost layer representing the nerve fiber layer contained a few capillaries. CtJrclS of endothelial cells. and isolat’edsmall glial cells. The pigment, epithelium (PE) was well developed at this stage. On day d a narww outer plexiform layer al~l~eareclin the posterior retina dividing the neuroblasts into outer and inner nuclear layers. By clrty i the outer plexiform layer had t?stmtle(~ to the IJra serrata thus cstdJhhirJg the basic laminar archit.ecture.

ddwlt. Details of the adult patt)ern of GS have heen clescril~etlpreviousIS- (Kiepe and Norenberg, 197i). Briefly. reaction product was present in Killer cell perikarp in the inner nuclear layer and in their processeswhich were traced through the plesifornl layers (Fig. 1). Theseprocessesterminated as funnels of react5on product in the ganglion cell layer and nerve fiber layer. and formed beads along the outer limiting nlembrane. The PE contained a trace amount of stain. Se&or)/. React.ion product was present,in the PE and was especially COIIS~~W~~I~IS anteriorly. No stain was evident in thr neural retina. Dny 1. Stain intensity increased slightly in the PF, along the anterior half of the retina (Figs 2 and 3), and dark perivascular stain was present in the nerve fiber layer near the optic nerve head (Figs 2 and 4).

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Thrys 2 -4. By day 2 reaction product ~3s ohservetl in the pcrikar\+a of isolated small glial cells in the ganglion cell layer in the posterior t,hree-fourths of the retina (Fig. I-). Increased numbers of stained glial cells and a more peripheral dist,rihutioll of ]wriva,scular stain in the nerve fiher layer were noted 1)~ dar 4. DU,I/S 5 Ned 6. Reaction product was first detected in a few bval t,o polygonal cells \I itll lwocesses in the inner nuclear layer in the posterior half of the retina on (lay 5 (Fig. r)). Identification of these cells as Miiller cells was substantiated by their position. r~tological characteristics and hv following t,heir (levelopmen~ during sulwqutwt da\-S. ti\+ day 6 n~any more stained Xiiller cell perika,rva were observed. Staill wan pr;wl)t ‘around hut not within ganglion cells presumaI;I~- repreSenting early Xiillrr cell ..i’llnnels”. l’erikarya of isolated small glial cells c‘ont’aining rcnct,ion pro(luct \vw~ ttvillwrt throughout the gnngliol~ ccl1 layer.

L)cr!/ 7. Miiller cell processeh in the post’erior half of the retina now contained reaction product8 which extended through the plexiform layers. outer nuclear layer. and formed funnels in the ganglion cell layer. f’erivascnlar stain was identified throughout the nerve fiber layer. Dnys 8 tr~tl 10. Reads of reaction product were located at the outer limiting membrane in the posterior half of the retina on day 8. Posteriorly. there was more stain in Killer cells (Figs 6 and i) where the pattern was similar to that found in t,he adult neura 1retina. T-‘,yday 10 this pattern encompassetl all hut the most ant.erior portion of ret,ina. Da!/ 12--Z ~~~orrths.Very little change occurred during this int~erval. Hy day 12 the tnnturc &in ])xttern had extentled to t.he retinal periphery (Fig. is). St,ain in the PE

GLUTAMISE

SYSTHETXSE

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FI(~. 2. IJay 1. Section demonstrates pr~pontlerance of stain in the retinal pigment cpithrlimnt anteriorly (arrows) and in the riliary body rpithelium (crossrtl arrnw). Stain in the inner layers of the retina near the optic nerre head (arrow heads) is in a peri\--awntar distrihutiorl as seen mow clmrt,v in Fig. 4. OS. optic nerve: T’. vitreous. 3u. FIG. 3. Higher magnification of periphery of day 1 retina shows pwmincnt stain in ttw pigmrnt epithelium (arrows) and lack of stain in the neural retina (NK). ‘.: 150. Fro. 4. Day 3. Retina shows reaction product around hkwl \-essrlr (~IWWS). anrt in a small glial cell (crosne(l ar~vw) in t hc ganglion cell layer. These ponitivelg st;lined glial rett. L: \!.PI‘C r,hservecl as early as day ‘7. SRI,, neuroblastic lager. i ?#I.

FIG. 9. Dav 3. Dark stain is present in the inner ciliary body in the poster& pigment epithelium of the iris (crossed arrow) t,oward the pupillary edge. C’. cornea; SK. neural retina. .‘75.

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Stain is dso noted of stain diminishes

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Cilitrry body. Dense reaction product was seenin the inner epithelium of the ciliary body in newborn rats (Fig. 9). In the youngest animals the intensity of stain equallecl that found in the peripheral portion of the retinal pigment epithelium. However, the intelrsc stain in the ciliary body was maintained into adulthoocl in contrast to the lossof stain in the PE observed in adults. Iris. Reaction product was present in Dheposterior ljigment epithelium of the iris from day 1 to adulthood (Fig. 9). Stain intensity was generally lessthan that in the ciliary body and no stain was observed toward the pupillary edge of the iris. Optic rterve. A slight to mockrate amount of reaction product was seen in all tlevelolm~ental st,agesfrom the newborn to adulthood. In young rats a diffuse pattern with accentuation in relation to glial-connective tissue septa was present [Fig. 10(a)]. In adults, the diffuse stain was no longer apparent’ [Fig. 10(b)]. Stain in the region of the nerve sheath was of moderate intensitv in developing rats and adults. Sections which included t#heoptic disc demonstrated a densedeposit’of react,ion product at, the imltx disc surfacr in Elschnig’s inner limiting nlem\Iran(~.

4. Discussion As establisheclby the immunohistochemical methods employed in this study, the development of GS in the neural retina beginscentrally and proceedsto the periphery. In the early postnatal period, this enzyme is locatetl in the ganglion cell layer in small glial cells and in a perivascular distribution. the latter presumably representing G8containing glial end-feet. GS is detected in a few Miiller cells as early as postnatal (lay 5. Hy day 7’ many Miiller cell perikarya contain reaction product and prominent E

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posit,ive Miillcr cell ~uwc~ssw itus nutetl itt the contml portiott ot’tttv wtitr;r. Ott 11;1y I:! tlip athtlt pattern is achievetl mitti tttc cwtiw rr:tiu;r slto\vittg lto\it,ivc> Xliith~t~ 1~~11 perikarp and processes. At t#his stag’ JIiillcr cells caoitt,aitt trtoqt of thrx (4s. Riochrniically. (3 levels are IoLv in the ttortrtat rat rc:t~itra until ]JOSbtlilt~ill tl;l!. !I (C’hader; 1971). From (lay 9 to 17 there is a. 25foltl itic:reasc it] ($S ;wtivit\- ~vltictt c!ecreases slightly afterwards. The lf)\v lt>vel of Gs detectetl Itictcllc~ttticil.tt\- tlrtt.itt,g thcl tlarlv tlevelolJnteilta1 period corre5pottlls with (3 irtttiti~itol~isttt~~tt~tiii~~~tl~ ti(~tttottstrat’ed in the PE and small glial cells in the ganglion cell la\-w. (bttr clat’tr itttlicatc~ t,hat the dramatic increase in hiochettlically clet~wtetl (23 act’i;?t,y OII th\-s 9 17 is a itt this Miiller cell phenon~enon. The slight’ly earlier increase cJf (2% t~elll~JllSt&~d study is pYJ)Jahly reflective rtf the high sensitivity of the ittLtttLt1LollistoCtt~~tltiGLt method. The slight decrease in sct)ivitv (JhSerVd after tlav 17 corrwjtottds wit~lt t’tica histochemicul decrease of GS in the Ph. The increase of 68 in the normal developing rat ret)ina C~JITeh~cS with crh\itl lna,turatiortal events especially in Miillw cells. The junctions Itetwen the apical r~ntls of' the Miiller cells and the photoreceptcJrs forming the outer limiting tt~wnhratw are cottipletctl on the 1Oth postnatal t lay (Kuwahara and Weidnian, 1974) tluri ng ;I t i ttw of marked increase of GS. On the same clay. Xiller Cc111 processrs arc fiJltrlc1 iLtl,jkCf?tlt to ltipolar cells in the inner nuclear lay. Ky day I2 synapt,ic compleses hsve fort~trtl in t’he inner plexiform layer and the first electroretinographic response (ERG) is tletected (Weidrnan and Kuwahara,? 1968). At this time histochernically dentonst,ratml GS is maximal and is found to he present in all Miiller cells. There is evidence to indicate that Miiller cells participate in the generation of part of t,he ERG (Karwoski and Proenza. 19’ii ; Miller. Dacheux and Procnza. 1977). The mechanism of the Miiller cell contrihut,ion t’o the ERG is not clear hut may IJC relat’ecl to changes in the extracellular pot,assium ion concentration resulting from synaptic activity (Mori. Miller and Tomi&. 1956; Karwoski and Proenza. 1977). (‘hanges in chxtracellular potassiunl ion concentration tna.y lte mediated t)y glutamate (Tori et al., 1976) which is a suttstratc for GS. Since the appearance of the E:RG corresponds with the initial tnaxintall,v demonstrated GS in XIiiller cells. the ERG may IIFLpart,lJ dependent on t,he full developnient of G8 in these cells. Increased GS activit,v can he S~KJWII in t)he rat retina from days 1 through 10 following the injectiori of triiotloth~roniiie and corticosteroitls on the first several days of life (Chader. 1971). Since the PE. Miiller cells and other glia contain C:S it cannot, be stated which cells are respnsible for t,his precocious increase of (:S act,ivity. Furt,her study is required to establish the site of this ]JhellcJlrlellOll. drr unexpected finding was the intense staining of the PE at, birth which \\-as niaintainerl to clay 12. tlitninishetl slight’ly by da!* I i. with only t,racc atttount,s left tty 2 months. The occurrence of GR in the PE is perhaps not surprising sittcc Miiller cells and the PE have a cotnniott Itrigirt from ncttroectodertil atitt a co~nmon fun&on of phagocvtosis (Hogan and Fccnev. 1963; c,g~. and Katsurne. 1970; Marntor. 1975). (:ti is colL~picLLous ill the PE at a d&e I\-hell it iS ;tlJsetlt (Jr nearly alJSeIlt frotlt ,\liiller cells. C,‘onsequently.the PE nmy ha\-c critical functions similar tu gliat cells such its the detoxification of anmtonia (Van clcn ISerg. 1970). during the periotl when Niiltct cells are ininlature. GS was also found in the inner cpit helium of the ciliary I~ody ant1 t,he posterior epit,heliunl of the iris in ycmng rat,s as IveIl as adults. There is evidence that these structures are concerned with the synthesis of the mucopolysaccharide component of the vitreous and aqueons humors (Rerrnan, 1964; Hogan, alvarado and Wecldell,

C:LUTAMINE

SYSTHETASE

IS

DET~ELOPJlES’l

197 1 ; Narmor: 1975). The action of GS in the ciliary 11c~ly ant1 iris gluta.n~int: required for mucopol~sRccharide smthesis.

143 could

lmvide

t’he

Hogan. Jl. .J., ~~lvarado. J. A. and \Veddell. J. E. (1971). (Xliary body and posterior chamlwr. lu /li.sfoloq,q of t//r HWIVM h’yr. I’. Wl. IV. B. Sanndrrs Co.. l’hi1adelphia. Hog;rtr. $1. .I. and Feeney. L. (19(G). Tl re ult~rastrhctiirc of rctirrnl rcsscls. I I I. Vascular-glial relationships. J. Ultmstruct. Ras. 9, 47-64. Karwoski. (1. J. and Proenza, L. XI. (1977). Relationship betxvcrn Mi~llcr cell rcsponscs. ;I local transrctinal potential, and pot,assinm flux. J. Nwrophysiol. 40, %4-5Y. Kcnncdp, .\. .J. Seal. JI. J. and Lolley. R. S. (l!l77). The distribution of amino acids within t 11~. rat ret,ina. J. Neurochern. 29, 157, !J. Kn~~abnra. T. and Weidman, ‘I’. .\. (1974). Development of the preii~ital rat retina. Iuw,s/. ~/~ht~t,ffl~tl~l.

13,

735-39.

~Iartncw, 11. F. (1975). Strnctnrc and function of the retinal pigments epithelium. Ijlt. ophth~r/l,nol. ( ‘lin. 15, 115SO. J1Qrtillc.z.Hernsndcz, A., Bell. K. P. and Sorenbcrg, >I. I). (1977). C+littamino spnthetasc: glial localization in brain. Science 195, 13568. Jlazlen. ft. C :., Mnrllenberg. C. G. and O’Brien, Y. ,J. (1970). L-( ~lnt~aminr wFructosr 6-phosphate atrridotransferase from bovine retina. Exp. Eye Rea. 9, l-l 1. .\lilkr. IZ. F.. Dacheiix, R. and Proenza, I,. (1977). Miiller ccl1 drpolarization evokrtl by antidroniic optic nerve stimulation. Brain Kr.s. 121, 16-6. Jlori. S.. Miller. \V. H. and Tomitw. T. (1976). Miiller cell function during spreading depression in frog retina. l’roc. Tot. Bend. Sri. V.S.S. 73, 1351-4. ~losc*or~;t, A. A. (1973). Induction of glntamine synthetase in embryonic ncnral retina: a mo&l for the regalation of specific gene espression in embryonic cells. In Biochemist,y oj (‘rll I~~~f~rwtinfion. (Eds Monroy. A. and Tsanrr. R.). Vol. ‘4. Pp. 1-S. ;\cademic, Press. Nrbv J’ork. .\losc~~in, AY .A. and Hubby, J. L. (1964). Experimentally induced cl~air~cs in plutanlotrilnsf~,r;\sc~ ac,tivity in embryonic tissnc. I)P~. Biol. 7, 19’SBOB. Sakaiic~. I’. K. and Kawaoi. A. #J. (1974). Perosidase-labeled antibody: ;I nen- method of (on. jllgat ion. J. Ilistoche~. C’ytocheuc. 22, 1084-91. Sxkn~rc, I’. K. and Pierce, G. B. *Jr. (1967). Enzyme-labeled ant,ibodies: Preparation and appli,~:r. t ion for the localization of antigens. J. Histochmn. Cytochwn. 14, !l79-31. Riclxh. R. E. and Norenberg. hl. D. (1977). Miiller cell localization of glritaminr syntlrctasc in nit, ret ina. ,Vtrtuw. Lomf. 268, 654---S. Rndrtick, D. and LVaelsch. H. (1955). Development of glntamotransferasc and glntamine synthctasr in the ncrvons system of the chick. J. Exp. Zoo/. 129, 309%26. Smit,h, C:. X. Jr. and Newsome, D. 11. (1978). The natnre and origin of the gl~rosaminogl~mrls of the embryonic chick vitreous body. I%~. Biol. 62, 65-77. Starr. $1. S. (1974). Evidence for the compartmentation of glatamatr nwtabolism in isolated rat rvt inn,. d. ,V~wochem. 23, 337-14.