Retinal Ganglion Cells Expressingthe FOS Protein After Light Stimulation

in the Syrian Hamster are RelativelyInsensitive to Neonatal Treatment

With Monosodium Glutamate

I. CHAMBILLE*Laboratoire de Physiologie Sensorielle, Institut National de la Recherche Agronomique,

78352 Jouy en Josas, Cedex, France

ABSTRACTIn nocturnal rodents, the c-fos gene is directly involved in the light mechanism of

resetting of the suprachiasmatic nucleus (circadian clock). Light also induces c-fos expressionin the retinal ganglion cell layer (GCL), but no attempt has been made to study the retinalresponses to the phase-shifting effects of light. The expression of the Fos protein in each of thetwo populations of the GCL (displaced amacrine cells [DACs] and ganglion cells [GCs]) wasanalyzed in hamsters after light stimulation delivered early (circadian time [CT13]) and in themiddle (CT18) of the subjective night. To evaluate as accurately as possible the number of GCsable to phase shift the locomotor activity rhythm (LAR), neonatal hamsters treated withmonosodium glutamate (MSG) were also used, an in vivo model which displays retinaldegeneration and LAR normally entrained by light. In nontreated hamsters, the number ofFos-immunoreactive (Fos-ir1) nuclei in the GCL was significantly higher at CT18 than atCT13. In MSG-treated hamsters, the number of Fos-ir1 nuclei was the same at both CTs andnonsignificantly different as those of nontreated hamsters at CT13. MSG treatment destroyedas many Fos-ir1 DACs as Fos-ir- DACs or Fos-ir1 GCs. Fos-ir1 GCs were less sensitive toneurotoxic than other GCs, as only 37% of them were destroyed by treatment versus 92% forFos-ir- GCs. At CT18, a maximum of 3,500 GCs expressed Fos protein in nontreated hamstersversus only 2,200 in MSG-treated hamsters. This minor subgroup was sufficiently potent tonormally synchronize the circadian rhythms to the Light/dark cycle in treated hamsters.J. Comp. Neurol. 392:458–467, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: circadian times; light stimulation; subjective night; displaced amacrine cells;

excitotoxicity model

Treatment of neonatal rodents with glutamate, an exci-tatory amino acid, or its by-product, monosodium gluta-mate (MSG), by intraperitoneal injection leads to acutedegeneration of the arcuate nucleus, inner layers of theretina, optic nerve, and visual pathways (Lucas and New-house, 1957; Cohen, 1967; Olney, 1969). Physiological andbehavioral abnormalities have been reported, such asobesity, sterility (Lamperti and Blaha, 1976; Tafelski andLamperti, 1977; Donham et al., 1990), loss of visualplacing response (Kizer et al., 1978), and defective discrimi-nation learning in a maze experiment (Pradhan andLynch, 1972). In a previous study conducted on Syrianhamsters, we showed that MSG treatment destroyed 56%of the cell populations of the ganglion cell layer (GCL),including 30% of displaced amacrine cells (DACs) and 89%

of ganglion cells (GCs; Chambille and Serviere, 1993).Only a subset of the surviving GCs was related to themajor components of the circadian timing system, i.e., thesuprachiasmatic nuclei of the hypothalamus (SCN), theintergeniculate leaflet (IGL), and the ventral part of thelateral geniculate nuclei (vLGN, Pickard et al., 1982;Chambille and Serviere, 1993). These surviving GCs are

Grant sponsor: INRA.*Correspondence to: Dr. Irene Chambille, Laboratoire de Physiologie

Sensorielle, Institut National de la Recherche Agronomique (INRA), CRJ-78352 Jouy en Josas, Cedex, France. E-mail: chambi@jouy.inra.fr

Received 3 December 1996; Revised 20 October 1997; Accepted 22October 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 392:458–467 (1998)

r 1998 WILEY-LISS, INC.

still able to mediate the photic entrainment of the circa-dian clock (Pickard et al., 1982; Miyabo et al., 1985;Rietvield et al., 1986; Chambille and Serviere, 1993). Theyphase shift locomotor activity rhythms (LAR) of animalskept in constant darkness (DD) when light pulses aredelivered at critical times of their circadian rhythms, i.e.,during the active phase of the LAR that corresponding tothe subjective night of animals (Chambille, 1997). Despitesevere retinal lesions due to treatment, we can thereforeconsider that the SCN and the photic entrainment path-ways via the retinohypothalamic tract (RHT) remainfunctional in MSG-treated animals.

In nocturnal rodents, a correlation between phase shiftsof the LAR produced by light pulses and induction ofseveral immediate early genes (IEGs), including c-fos, hasbeen shown in SCN (Kornhauser et al., 1990; Rusak et al.,1990, 1992; Sutin and Kilduff, 1992). IEGs are rapidly andtransiently expressed in the SCN cells only when lightpulses phase shift the rhythm (Kornhauser et al., 1990;Ebling et al., 1991; Chambille et al., 1993). Expression ofthe Fos protein is phase-dependent, because the highestnumber of Fos-immunoreactive (Fos-ir1) cells in the SCNis observed when light stimulation is given in the middle ofthe subjective night (Rea, 1992; Earnest et al., 1992;Chambille et al., 1993). Photic induction of the c-fos genewas also reported in retinal neurons of rodents (Sagar andSharp, 1990; Earnest et al., 1990; Koistinaho and Sagar,1995). In the Syrian hamster, kept in DD, a strongerexpression of the Fos protein was induced in the ganglioncell layer (GCL) when light stimulation was deliveredduring the active phase of the LAR (subjective night) thanduring their inactive phase (subjective day; Chambille etal., 1993).

Numerous data suggest that the c-fos gene is involved inthe photic mechanism which resets the circadian clock(Schwartz et al., 1995) and that Fos protein expression is agood marker of activation of light-responsive cells of thecircadian system in both the retina and SCN.

The aim of the present study was to quantify the cells ofthe GCL expressing the Fos protein in the phase shiftingresponses to light. Investigations were performed at twotimes of the animal’s circadian cycle (circadian times, CTs)at which light causes maximal phase shifts in oppositedirections, i.e., early in the subjective night (delay phase,CT13; by convention, CT12 represents the onset of thesubjective night of nocturnal animals or the onset of theirLAR) and in the middle of the night (phase advance,CT18). To evaluate as accurately as possible the popula-tion of ganglion cells able to phase shift LAR, the in vivomodel of MSG excitotoxicity was also used. The study ofFos expression in the retina of control hamsters andMSG-treated hamsters led us to analyze the neurotoxiceffects of glutamate on two cell populations of the GCL(DACs and GCs) whether or not they express the Fosprotein.

The present results show that: (1) the ganglion cellsexpressing the Fos protein (Fos-ir1 GCs) were less sensi-tive to the neurotoxic effect of glutamate than other GCs;(2) MSG treatment destroyed as many Fos-ir1 DACs asDACs not expressing the protein (Fos-ir- DACs); (3) amaximum of 3,500 GCs expressed the Fos protein innontreated hamsters at CT18; and (4) Only 2,200 Fos-ir1

GCs were counted in the retina of MSG-treated hamstersafter light stimulation at CT18, normally phase-shiftinganimals. These results were presented at the Conference

on ‘‘Circadian Light and Regulation’’ held in Lyon, France,May 1996.

MATERIALS AND METHODS

Animals and treatments

Animals. All animal experiments were carried out inaccordance with the European Communities Council Direc-tive of 24 November 1986 (86/609/EEC).

The hamsters were born in the laboratory colony andmaintained under a light/dark (LD) cycle of 16:8 hours at aroom temperature of 23 6 2°C. On the day of birth (D1),newborn males were transferred with their mother intoventilated boxes under controlled lighting conditions con-sisting of a 12:12 LD cycle. Food and water were given adlibitum.

MSG treatment. Treatment was described in a previ-ous paper (Chambille and Serviere, 1993). Briefly, 5-day-old males received a first intraperitoneal (i.p.) injection of5 mg/g of body weight of monosodium glutamate (Merck;Darmstadt; Germany) dissolved in 0.1 ml of saline, fol-lowed by daily injections increasing from 5 to 8 mg/g fromD6 to D10. Control males (one per litter) received dailyinjections of saline from D5 to D10 or were not injected.

Groups and surgical procedure. After weaning, theanimals were separated into two groups: one group ofcontrol hamsters and one group of MSG-treated hamsters.Two to 3 months later, half of the animals of each groupwas selected for optic nerve degeneration experiments.The animals of these two subgroups were deeply anesthe-tized by i.p. sodium pentobarbital (Sanofi, Libourne,France). The orbital cavity of one eye was opened and thedorsal muscles were detached from the eyeball. The opticnerve was carefully separated from the ophthalmic arterybefore complete transection. A piece of gelfoam soakedwith sterile saline was then applied to the stump and theorbital cavity was sutured. Fos experiments were com-menced 3 months later, because no GCs survived in theGCL after this period, as previously shown (Chambille andServiere, 1993).

Fos expression experiments

Four 5-month-old MSG-treated hamsters and controlhamsters, with or without sectioned optic nerve (ON),were individually housed in cages equipped with runningwheels. LAR was recorded for 2 weeks under the same12:12 LD cycle as previously, then for 2 weeks under DD.

Light stimulation. Only hamsters generating a ro-bust free-running LAR and clear onset of the active phasewere included in the study. Fos expression was induced attwo circadian times of the LAR, i.e., early in the subjectivenight (CT13) and in the middle (CT18) of the subjectivenight. These two CTs were chosen because they are knownto cause the greatest phase shifts of LAR and maximal Fosexpression in the retina and SCN. Light stimulationconsisted of sequences of 30 flashes (200 ms each) at afrequency of 6 per minute, as previously described (Cham-bille and Serviere, 1993).

Immunohistochemistry of the retina. One hour afterlight stimulation, the animals were deeply anesthetizedwith an overdose of pentobarbital under dim red illumina-tion. The nasal corner of the cornea was marked with blackink to allow orientation of the retina. Hamsters were thenperfused transcardially with 200 ml of 37°C saline solutioncontaining 1% sodium nitrite, followed by 300 ml of 4%

FOS-EXPRESSION IN LESIONED RETINAE BY GLUTAMATE 459

paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB).The eyes were removed immediately, the anterior chamberwas opened to remove the lens, and the eyeballs werepostfixed for 4 hours in the same fixative at 4°C. Theretinae were washed overnight in PB and the vitreoushumor was removed.

Floating retinae were treated for 30 minutes at roomtemperature in 0.1% hydrogen peroxide, carefully washedand then incubated in a blocking solution of 10% normalgoat serum in phosphate-buffered saline PBS (UnipathLTD, Basingstoke, England) with 0.3% Triton X-100 (NG-PBST). They were then incubated at 4°C for 48-72 hours inanti-c-fos primary antibody (Oncogene Science; Uniondale,NY) diluted to 1:10,000 in 1% NGPBST, rinsed three timesin 1% NGPBST and incubated in biotinylated goat anti-rabbit IgG diluted 1:500 (Byosis SA, Compiegne, France)in 1% NGPBST overnight. After several rinses in PBS, theretinae were incubated for 1 hour in (ABC) reagentVectastain kit (Vector Laboratories, Burlingame, CA),then for 5 to 10 minutes in a chromogen solution consistingof 0.02% 3,3’-diaminobenzidine tetrahydrochloride (DAB),0.3% nickel ammonium sulfate in PBS and 0.035% hydro-gen peroxide. Free retinae were mounted on gelatin-coatedslides with the ganglion cell layer facing upwards, andwere dehydrated and coverslipped in Depex.

Isodensity maps and Fos-ir nuclei counts

Isodensity maps of the Fos-immunoreactive (Fos-ir1)nuclei were obtained from retinal wholemounts using aBiocom 500 image analyzer. The whole retina was scannedby moving a 0.04 mm2 square grid at 500-µm intervals.Fos-ir1 nuclei were counted in each square and the num-ber was expressed as a density (nuclei/mm2) which wasplotted on the map according to XY coordinates. Isodensitylines were then drawn from each plot.

The total number of Fos-ir1 nuclei was estimated fromthe number of labeled nuclei in the samples and expressedfor the whole retinal surface. The same protocol wasapplied to normal and axotomized retinae. In these axoto-mized retinae, the number of Fos-ir1 nuclei representedonly Fos-ir1 DACs, since all CGs were destroyed duringthe 3 months following section of the ON. The number ofFos-ir1 GCs was therefore estimated by the differencebetween the total number of Fos-ir1 nuclei counted inintact retinae (DACs and GCs) and the number of Fos-ir1

nuclei counted in axotomized retinae.Two-way analyses of variance (ANOVA, generalized

linear model, Systat for Windows, version 5, Evanston, IL,1992) was performed to test: (1) the effects of MSGtreatment and of circadian times, and (2) the effects oftreatment and of section of optic nerve on light-inducedretinal Fos expression. Each analysis was followed byTukey’s post-hoc test for to assess differences amonggroups.

RESULTS

Fos expression in the GCL of controlhamsters and MSG-treated hamsters with

intact optic nerves

In this paper, the term ‘‘control hamsters’’ was reservedto nontreated hamsters with intact ON. Other animalswere either nontreated hamsters with sectioned ON orMSG-treated hamsters with intact ON or sectioned ON.

In our previous study (Chambille and Serviere, 1993),we have shown that there were 60% of DACs, varying insize from 5 to 15 µm, and 40% of GCs (7 to 21 µm) in GCL.

Control hamsters. Light exposure during the subjec-tive night induced the expression of Fos protein in thenuclei of GCL. Small and large Fos-ir1 nuclei were ob-served, which suggests that the reaction product could beexpressed in the two different cell types: DACs and GCs.Fos-ir1 nuclei were scattered over the entire retina (Fig.1A) with a higher density in the horizontal streak than inthe periphery. The mean number of Fos-ir1 nuclei was12,602 when light was applied at CT18 and 10,187 forstimulation at CT13 (Table 1). Light therefore inducedsignificantly more Fos expression in GCL at CT18 than atCT13 (difference 19%; P , 0.02; first ANOVA).

MSG-treated hamsters. Fos immunoreactivity wasclearly detected in the GCL, and labeled nuclei were stilldistributed over the entire retina (Fig. 1C). A significantreduction in the number of Fos-ir1 nuclei was observed atCT18 (P , 10-3, first ANOVA, Table 1). In contrast tocontrol hamsters, the mean numbers of Fos-ir1 nuclei wereidentical after light stimulations at CT13 and CT18 (Table1); these numbers were not significantly different fromcells expressing Fos protein in retina of nontreated ham-sters at CT13 (first ANOVA, Table 1). These resultssuggest: (1) that all the surviving cells capable of express-ing the Fos protein in MSG-treated hamsters were acti-vated during the subjective night by light stimulation,regardless of the time of exposure, and (2) MSG treatmentdestroyed Fos-ir1 cells equipotent to subpopulation express-ing protein Fos only at CT18 in nontreated hamsters. Thispool represented 67% of the maximal population of Fos-ir1

cells observed in control hamsters at the same time(Table 1). Finally, MSG treatment destroyed about 4,130potentially Fos-ir1 cells, i.e., one-third of the total Fos-ir1

population of the retinal GCL (Table 1).

Fos expression in the GCL of nontreated andMSG-treated hamsters with sectioned

optic nerves

In our previous study, DACs were directly quantifiedthree months after sectioning the ON and the populationof GCs was estimated by the difference between the totalcell population in the GCL of intact retinae and that ofDACs in axotomized retinae. The same protocol wasapplied in the present study. The number of Fos-ir1 GCswas estimated from the total population of nuclei express-ing the Fos protein at CT18 in intact retinae and that ofFos-ir1 DACs counted at CT18 in axotomized retinae. Cellcounts were performed only at CT18, as this time corre-sponded to the maximal level of Fos expression in controlhamsters and because this expression was similar at bothCTs in MSG-treated hamsters.

In the axotomized retinae of nontreated hamsters andMSG-treated hamsters, Fos-ir1 nuclei were still scatteredover the entire surface of the ganglion cell layer, suggest-ing important activation of the DAC population (Fig.1B,D). Their Fos-ir1 DAC population were respectivelysignificantly reduced (second ANOVA) since evaluated tobe 9,093 in nontreated hamsters (i.e., 72% of the totalnumber of Fos-ir1 nuclei of the GCL of control hamsters;Table 1, P , 10-3) and 6,273 in MSG-treated hamsters (i.e.,74% of the total number of Fos-ir1 nuclei of the GCL ofMSG-treated hamsters; Table 1, P , 0.05). About 3,500Fos-ir1 GCs were therefore detected in nontreated ham-

460 I. CHAMBILLE

sters (28% of the total population of Fos-ir1 nuclei incontrol hamsters, Table 1) versus about 2,200 Fos-ir1 GCsin the treated model (26% of the total population of Fos-ir1

nuclei in MSG-treated hamsters, Table 1). It is worthnoting that the Fos-ir1 DACs and Fos-ir1 GCs surviving

after MSG treatment were in the same proportion as innontreated hamsters, which could mean that the potencyof the neurotoxic effect of MSG is similar on both Fos-ir1

populations of the ganglion cell layer. Finally, treatmentwith glutamate destroyed 2,820 Fos-ir1 nuclei belonging tothe DAC population and consequently 1,310 potentiallyFos-ir1 GCs (Table 1).

Neurotoxicity of glutamate on the retinalganglion cell layer

Table 2 summarizes the neurotoxic effect of MSG on theretinal GCL. Some of these data are derived from aprevious study in which the decrease in DACs and GCs inthe GCL was estimated in MSG-treated animals (Cham-bille and Serviere, 1993). In Table 2, DACs and GCs ofnontreated hamsters and MSG-treated hamsters weresubdivided into two groups: cells activated by a lightstimulus at CT18 (Fos-ir1 cells) and cells remaining silent(Fos-ir- cells).

In nontreated hamsters, the Fos-ir1 to Fos-ir- ratio was1:9 for DACs (9,093/84,366) and 1:17 for GCs (3,509/58,217; Table 2). In MSG-treated hamsters, this Fos-ir1 toFos-ir- ratio was always 1:9 for DACs (6,273/58,887), butwas much higher for GCs, up to 1:2 (2,199/4,477; Table 2).The mortality was identical in Fos-ir1 and Fos-ir- DACs(31% and 30%), whereas a marked difference was observedbetween the mortality of Fos-ir1 and Fos-ir- GCs (37%versus 92%; Table 2). Fos-ir1 cells were therefore much

TABLE 1. Fos Protein Expression in the Retinal GanglionCell Layer of Nontreated Hamsters and Monosodium Glutamate

(MSG)-Treated Hamsters1

CT13Intact

optic nerveFos-ir1

DACs 1 GCs

CT18

Intactoptic nerve

Fos-ir1

DACs 1 GCs

Sectionedoptic nerve

Fos-ir1

DACs

Estimationof the

Fos-ir1

GCs

Nontreatedhamsters 10,187 6 5872 12,602 6 6233 9,093 6 3816 3,509

Mean 6 S.E.M.(number) (n 5 6) (n 5 6) (72%) (n 5 5) (28%)

MSG-treatedhamsters 8,879 6 1964 8,472 6 4385 6,273 6 5217 2,199

Mean 6 S.E.M.(number) (n 5 6) (n 5 7) (74%) (n 5 6) (26%)

Number of deadFos-ir1 cells 4,130 2,820 1,310

1The mean number of Fos-ir1 nuclei/retina was estimated after light stimulation atcircadian times (CTs) 13 and 18. In retinae with intact optic nerve (ON), Fos-ir1 nucleicorrespond to the Fos-ir1 displaced amacrine cell (DAC) and Fos-ir1 ganglion cell (GC)populations. In retinae with sectioned ON, Fos-ir1 nuclei only represent the Fos-ir1

DAC populations because all GCs are destroyed after 3 months of optic nerve section.Two two-way analyses of variance following by Tukey’s test were performed to analyzedata. 2 versus 3 (P , 0.02); 3 versus 5 (P , 1023); 3 versus 6 (P , 6.1023); 5 versus 7(P , 0.05); 2, 4, and 5: not significantly different.

Fig. 1. Fos immunostaining in flat-mounted retinae of (A,B) non-treated hamsters and (C,D) monosodium glutamate (MSG)-treatedhamsters after light stimulation at circadian time (CT)18. A: Non-treated hamsters with intact optic nerve (ON); B: Nontreated ham-

sters with sectioned ON; C: MSG-treated hamsters with intact ON;D: MSG-treated hamsters with sectioned ON. A,C Fos-ir1 nuclei corre-spond to Fos-ir1 displaced amacrine cells (DACs) and Fos-ir1 ganglioncells (GCs); B, D only represent the Fos-ir1 DACs. Scale bar 5 150 µm.

FOS-EXPRESSION IN LESIONED RETINAE BY GLUTAMATE 461

less sensitive to MSG toxicity; thus, Fos-ir1 GCs andFos-ir1 DACs were in a similar proportion in nontreated(3,509/9,093; i.e., 1:2.6) and MSG-treated hamsters (2,199/6,273; i.e., 1:2.9; Table 2).

Spatial distribution of Fos-ir1 populations ofthe GCL in nontreated hamsters and

MSG-treated hamsters

In control hamsters (with intact ON), the regionaldistribution of Fos-ir1 nuclei reflected the spatial organiza-tion of the total cell population reported in our previousstudy (Chambille and Serviere, 1993). Fos-ir1 nuclei werescattered all over the retina with the highest cell density inthe inferior hemiretina. In the inferior temporal sector,Fos-ir1 nuclei were most numerous in a crescent-shaped

region located near the horizontal meridian, where densi-ties exceeded 500 per mm2 (1, Fig. 2A) The position of thisarea centralis was similar to that of the total population,but the cell densities were 10 times lower. Three circum-scribed zones, characterized by a gradual decrease of celldensity from 400 cells/mm2 to 100 cells/mm2, were ob-served around this central area. The decrease was slowerin the inferior part than in the superior part of the retinaand lower in the temporal than in the nasal sector. Nearthe outer margin of the retina, the cell density continued todecline to reach less than 100 cells/mm2 (Fig. 2A).

In nontreated hamsters with axotomized retina, Fos-ir1

nuclei were less numerous, as only Fos-positive DACswere still present. The position and cell density in the areacentralis were similar to those in the normal retina.

TABLE 2. Comparison of the Neurotoxic Effect of MSG Treatment on the Retinal Fos-ir1 DAC and GC Populationsand the Fos-ir2 DAC and GC Populations1

Total population2 Fos-ir1 population Fos-ir2 population

Counted DACs Estimated GCs Counted DACs Estimated GCs Counted DACs Estimated GCs

Nontreated hamstersMean 6 S.E.M. 93,459 6 3,184 61,726 9,093 6 381 3,509 84,366 58,217

MSG hamstersMean 6 S.E.M. 65,160 6 2,215 6,676 6,273 6 521 2,199 58,887 4,477

Mortality (%) 30 89 31 37 30 92

1The Fos-ir1 populations represent the highest responses of DACs and GCs, i.e., the Fos protein expression after light stimulation at CT18.2Total population of DACs and GCs (two left columns) are derived from Chambille and Serviere (1993).

Fig. 2. Isodensity maps of the distribution of the Fos-ir1 populations (Fos-ir1 DACs and Fos-ir1 GCs)in intact retinae of nontreated hamsters (A) and monosodium glutamate (MSG)-treated hamsters (C) andof Fos-ir1 DAC population in axotomized retinae of nontreated hamsters (B) and MSG-treated hamsters (D).

462 I. CHAMBILLE

Around the area centralis, two large circumscribed regionsof decreasing densities were recorded with densities lowerthan those of regions 2 and 3 of the controls, suggestingthat the dead potentially Fos-ir1 GCs were evenly distrib-uted in these regions (Fig. 2B).

In MSG-treated hamsters with intact ON, the Fos-ir1

nuclei of surviving cells were organized in a more simplepattern than in control hamsters. Only four zones ofdecreasing densities were observed, while the fourth zonewas limited to the border of the retina. The highest celldensities were found in two circumscribed regions of theinferior temporal retina: the central region was small,similar to the area centralis of control hamsters with celldensities ranging from 400 to 500 cells/mm2; the intermedi-ate region had a ventrolateral extension with 300 to 400cells/mm2. The third region displayed little or no regionalvariations, because Fos-ir1 nuclei were regularly scatteredwith densities ranging between 100 and 300 cells/mm2

(Fig. 2C).In MSG-treated hamsters with sectioned ON, the topog-

raphy of the surviving Fos-ir1 DACs still showed a re-gional distribution, suggesting that the cells remainedorganized (Fig. 2D). The area centralis (with densitieshigher than 400 cells/mm2), its satellite ring and itsventrolateral extension were still present, but their sur-face areas were reduced. The region corresponding to thethird region of the intact retina of treated hamsters (i.e.,with cell densities ranging from 100 to 300 cells/mm2) wasalso restricted to a large streak elongated along thenasotemporal axis. Since the streak ran just dorsally to theoptic disk, the inferior hemiretina still showed the highestcell densities. A homogenous distribution of cells withdensities lower than 100 cells per mm2 was observed in thesuperior retina.

DISCUSSION

The results obtained in the present study clearly demon-strate that MSG treatment did not prevent Fos proteinexpression in the retina of light-stimulated animals. Theyprovide a further example of the specific insensitivity of asubset of retinal GCs to MSG.

Retinal Fos immunoreactivity and thecircadian system

Since 1989, many studies have shown a light-dependentphase activation of the c-fos gene in the SCN of rodents. Infree-running animals maintained in DD, Fos protein ex-pression can be induced in the SCN during the subjectivenight (Rea, 1989) only at CTs when light has phase-shifting effects (Kornhauser et al., 1990; Rea, 1992; Cham-bille and Serviere, 1993).

In the retina, an induction by light of c-fos gene has beenreported in amacrine cells of the inner nuclear layer (INL)and in cells of the GCL depending on environmentallighting conditions. In rodents, c-fos mRNA was observedduring exposure to constant illumination or immediatelyfollowing the onset of the light period of an L/D cycle(Earnest et al., 1990; Gudehithlu et al., 1993). In animalsmaintained in DD, Fos protein was induced only after alight stimulation (Sagar and Sharp, 1990; Koistinaho etal., 1993; Chambille et al., 1993; Koistinaho and Sagar,1995); the expression was not similar between the subjec-tive day and the subjective night (Chambille et al., 1993). Adifferential expression of c-fos between retinal populations

has also been hypothesized (Nir and Agarwal, 1993) andreported between outer nuclear layer (ONL) and INL orGCL (Yoshida et al., 1993). In animals kept in DD, arhythmic expression of c-fos mRNA was described in theONL, suggesting an endogenously generated rhythm(Yoshida et al., 1993) which could depend on cyclic fluctua-tions of an oscillator located in the photoreceptor layer orin the ONL. In fact, the presence of a circadian oscillatorregulating melatonin synthesis has been recently demon-strated in vivo in the hamster (Tosini and Menaker, 1996).

This study provides, in our lighting conditions, a quanti-tative estimation of the highest levels of photic inductionin the GCL at the two CTs when light phase-shifts theLAR. Light stimuli used here were a sequence of 30 flashes(200 ms each) at a frequency of 6 per minute. Theyproduced, in nontreated hamsters, robust phase shifts ofLAR at CT13 (89 minutes, 8 animals) and CT18 (130minutes, 12 animals; Chambille, 1996). Similarly, meandelay and advance of the active phase of the LAR were notsignificantly affected by MSG treatment (Chambille, 1997).Light stimuli used in the literature to obtain phaseresponse curves are continuous with durations rangingfrom 1 second to several hours (Johnson, 1990). For a givenwavelenghth of light (maximum sensitivity 503 nm), theamplitude of the phase shift is related only to the numberof photons whatever the duration of the pulse, up to 45minutes (Takahashi et al., 1984). In our case, the intenseand discontinuous stimulus induced behavioral delay andadvance phase shifts with amplitudes as large as those ofthe phase response curve reported by Takahashi et al.(1984) for 60 minutes and more white light pulses given atthe same circadian times in the same species. In thehamster SCN, the c-fos mRNA levels at CT19 continue toincrease at an irradiance of 1014 photons cm-2 s-1 (durationof pulse 5 minutes), whereas the phase shift of LARsaturates at approximately 110 minutes for an irradianceof 1013 photons cm-2 s-1 (Kornhauser et al., 1990). Thus, wecan consider that the light stimulation used in this studyproduced maximal behavioral phase shifts and retinal Fosprotein expression near saturation.

Since the Fos-ir1 GC population is a minority of surviv-ing GCs, it is not possible to be sure that it is not thepopulation of ganglion cells that do not express Fos inresponse to light which mediates photic entrainment. Theopposite is not also directly demonstrated, but manyfeatures suggest that retinal Fos-ir1 cells are probablyinvolved in photic entrainment of a central circadian clock:(1) there is evidence for circadian control in the rat eye ofphotoreceptor disc shedding (Korenbrot and Fernald, 1989);(2) there is a clock in the retina (Tosini and Menaker,1996); (3) circadian and nycthemeral c-fos mRNA rhythmsare present in the ONL of retina (Yoshida et al., 1993); asin SCN (4) a Fos immunoreactivity is observed in GCL ofdark-adapted retinae only after light stimulations; (5) Fosimmunoreactivity is stronger for a light stimulation deliv-ered during subjective night than during subjective day(Chambille et al., 1993); (6) strong retinal Fos immunoreac-tivity is associated with phase shifts of free-running LAR.We do not know how this retinal oscillator operates(autonomously or driven by SCN) but it is evident that itposseses some functional characteristics of the centralcircadian pacemaker. Thus, if we accept that c-fos expres-sion is a reliable marker of cell activation by light in theretina as well as in the SCN, we can consider that Fos-ir1

cells observed in retina of normal hamsters represent the

FOS-EXPRESSION IN LESIONED RETINAE BY GLUTAMATE 463

cell population mediating the reset by light of circadiansystem.

In nontreated hamsters, only 3,500 GCs were Fos-ir1

after light stimulation at CT18. These cells are morenumerous than retinal GCs identified by Pickard (1980,1982) in the hamster using retrograde transport of horse-radish peroxidase (HRP) injected into SCN. However, didall retinal terminals take up the tracer, and transport it?Was there enzymatic degradation of HRP in GCs? In anearlier study using transneuronal transport of the Barthastrain of herpes virus in the rat, a more sensitive labelingthan free HRP, Moore et al. (1995) showed that the subsetof GCs projecting onto the SCN via the retinohypothalamictract (RHT) is much larger than the population of afferentsto the SCN generally referred to in the literature on thebasis of the study by Mason and Lincoln (1976). Unfortu-nately, no estimation of the number of the GCs giving riseto RHT was reported. Thus, in rodents, these GCs werenever exactly quantified because of large technical difficul-ties in the tracing experiments from SCN, variable sensi-tivity of tracers and their detection systems. Finally, theganglion cells expressing the Fos protein probably give amaximal estimation of the pool of retinal componentsbelonging to the circadian timing system. In this pool, inaddition to the GCs projecting directly onto the SCN, areincluded other GCs innervating surrounding areas such asthe anteroventral periventricular nucleus, the lateral hy-pothalamic area, and the ventral part of paraventricularnuclei, where direct retinal projections (Johnson et al.,1988) and photic induction of Fos protein have also beenobserved (Rusak et al., 1990; Chambille et al., 1993;Teclemariam-Mesbah et al., 1995). It also contains cellsthat directly innervate the IGL and some GCs projectingonto both IGL and SCN via collateral fibers of the retinohy-pothalamic tract (Pickard, 1985; Treep et al., 1995). In fact,Fos-ir1 nuclei have been observed in light/dark-main-tained rats (Aronin et al., 1990) or in Syrian hamstersafter light stimulation (Ebling et al., 1991; Janik andMrosovsky, 1992) in the IGL and vLGN, i.e., two regionsinvolved in photic regulation of circadian rhythms via thegeniculohypothalamic tract (Harrington and Rusak,1987;Pickard et al., 1987; Pickard, 1989). As Moore et al. (1995)did not provide any quantitative estimation of the GCsgiving rise to retinohypothalamic projections, it is difficultto compare the data reported in this paper with those ofthis previous study and to conclude that Fos-ir1 GCsrepresent the same population as GCs labeled by transneu-ronal transport of herpes virus. Moreover, the two studieswere conducted on two different species showing a differ-ent rostrocaudal pattern of retinal projections onto theSCN (Miller et al., 1996). Nevertheless, the subsets of GCsidentified in the two studies had a similar spatial distribu-tion over the entire retina, with slightly greater celldensities in the temporal hemiretina. Therefore, Fos-ir1

GCs could give an estimation of maximal population ofGCs that mediate photic synchronization of circadianrhythms.

In MSG-treated hamsters, the circadian system remainsfunctional because free-running LAR in DD is still trig-gered by a light/dark cycle (Pickard et al., 1982; Chambilleand Serviere, 1993) and can be phase-shifted by light atCT13 and CT18 with the same amplitude as in nontreatedhamsters (Chambille, 1997). Light also induced Fos immu-noreactivity in the GCL, but Fos-ir1 cells were signifi-cantly less numerous than in control hamsters at CT18.

Dead Fos-ir1 GCs were probably totally involved in thephotic entrainment of the circadian clock. Three observa-tions can account for such a statement: (1) there is areduction in the volume of labeled retinal terminals in theSCN (Chambille and Serviere, 1993); (2) there is a signifi-cant decrease in Fos immunoreactivity in the IGL, vLGNand, to a lesser extent, SCN of MSG-treated rats (Edel-stein et al., 1995); and (3) there is a 20% reduction of thenumber of nuclei expressing Fos protein at CT18 in theSCN of MSG-treated hamsters (personal observations).Finally, in MSG-treated hamsters, only 2,200 Fos-ir1 GCssurvived after treatment (Table 2). They constitute asubgroup sufficiently potent to convey the light informa-tion to the SCN and to normally synchronize the circadianrhythms to the light/dark cycle.

In MSG-treated hamsters, Fos-ir1 cells (DACs and GCs)are in the same number at both CTs. It is possible,although not demonstrated, that cells expressing Fosprotein at CT13 and CT18 in MSG-treated hamsters andat CT13 in control hamsters are the same cells. Neverthe-less in nontreated hamsters, an additional subpopulation(19% of total population) expressed Fos only at CT18. Thisobservation suggests that Fos-ir1 GCs could be morenumerous at CT18 than CT13. In the SCN of controlhamsters, Fos immunoreactivity is also higher at CT18than CT13 and expressed in different regional cell subsets(Rea, 1992; Chambille et al., 1993; Romijn et al., 1996).One wonders whether such regional variations are stillpresent in the SCN of MSG-treated hamsters in whichafferent information seems to be independent of the CT ofstimulation, a situation similar to surgical isolation ofSCN electrically stimulated via the sectioned optic nerve(Earnest and Olschowka, 1993). Spatiotemporal cyclicchanges of c-fos expression could therefore reflect cyclicfluctuations of SCN intrinsic conditions. Studies are inprogress to test this hypothesis.

In MSG-treated hamsters as in control hamsters, therewas one Fos-ir1 DAC out of 9 DACs, since mortality wassimilar for all DACs (Fos-ir1 and Fos-ir-) but one Fos-ir1

GC out of 2 GCs due to the relative insensitivity of Fos-ir1

GCs to MSG. The loss of cells affected the entire surface ofthe retina, but surviving Fos-ir1 cells remained organizedand retained certain spatial characteristics of their distri-bution, such as the area centralis in the ventrotemporalsector and its satellite crescent. Finally, Fos-ir1 GCs werelikely distributed all over the retina because cell densitieswere altered in every part of the retina after section of theON. Can we suppose that Fos-ir1 DACs and GCs areorganized in a network of interconnected neurons asrecently described by Nirenberg and Meister (1997) forlight response of some GCs in mouse’s retina?

Neurotoxicity of MSG

In this model of MSG neurotoxicity, GCs were muchmore sensitive to neonatal MSG treatment than DACs. Inour previous study, using two types of injections overdifferent periods of time, we demonstrated that 89% of GCswere regularly killed versus 30% of DACs (Chambille andServiere, 1993). Apart from this MSG model, anothermodel of toxicity based on intraocular administration ofhigh doses of N-methyl-D-Aspartate (NMDA), a glutamateagonist selective for the NMDA subclass of receptors,induced similar severe retinal lesions of the adult ratretina (Silipandri et al., 1992). Using this model of excito-toxicity, Sabel et al. (1995) recently observed a dose-

464 I. CHAMBILLE

dependent loss of GCs, a neuroprotective effect of MK801(a noncompetitive NMDA antagonist) and a high correla-tion between the number of surviving GCs and the visualperformances of rats immediately after the lesion. In fact,the animals showed a great capacity to compensate forimpairment of behavioral tasks, since they were able toovercome their functional deficits within about 2-3 weeksin spite of only 13% of surviving GCs. Intraperitonealinjections of MSG in neonatal hamsters or intraocularadministration of high doses of NMDA in adult ratstherefore seem to produce two similar models endowedwith only 11-13% of GCs connected to their target cells.

The present results show that only 37% of the Fos-ir1

GCs died after MSG treatment, whereas 92% of Fos-ir-

GCs disappeared. Retinal GCs that display Fos inductionto a light stimulus therefore appear less sensitive thanother cells to the neurotoxic effects of MSG. However, themechanism(s) by which these cells are protected in vivoagainst the toxicity of MSG have not yet been elucidated.Several hypotheses can be proposed: a system involvingNMDA receptors, production of nitric oxide (NO), and anegative feedback loop. NMDA receptors have often beenconsidered to be the main receptors involved in glutamateneurotoxicity in brain and retina (Akaike et al., 1994).AMPA/kainate receptors could also be involved in thistoxicity; however, they are rapidly desensitized to glutamicacid, and many of their subunits do not allow the passageof calcium ions. (Doble, 1995). Two cytotoxic cascades maybe involved in the retinal cell mortality of MSG-treatedhamsters. The first one would be due to overstimulation ofan NMDA receptor, an increase in intracellular Ca21

(Garthwaite, 1989; Meldrum and Garthwaite, 1990;Farooqui and Horrocks, 1991), activation of calcium-dependent proteases, phospholipases and endonucleasesleading to neuronal death (Doble, 1995; Weber et al.,1995). The second cascade would involve the reactivefree-radical NO, synthesized by NO synthetase (NOS), akey agent in NMDA receptor-mediated toxicity (Bredt etal., 1990; Kiedrowski et al., 1992; Lipton et al., 1993). Athigh concentrations, the gas easily permeates the cellmembrane, diffusing into adjacent cells and acting as atoxin for neighboring neurons. However, NOS-containingneurons may possess a protection system against NO-mediated toxicity and may therefore be relatively pre-served (Gartwaite, 1991; Dawson et al., 1991; Bredt et al.,1991; Bruhwyler et al., 1993). It has been demonstrated invitro that the endogenously produced NO (like other freeradicals) could be responsible for sustained inhibition ofNMDA receptors (Manzoni et al., 1992) by the direct actionat the redox modulator site of the NMDA receptor-channelcomplex (Aizenman et al., 1990; Lei et al., 1992). SurvivingFos-ir1 DACs and Fos-ir1 GCs might therefore have beenspecifically protected from the excess glutamate by amechanism involving this type of negative feedback loop.The presence of NOS has been indirectly demonstrated inthe INL (Sandell, 1985; Sagar, 1986; Vaney and Young,1988; Mitrofanis, 1989) and GCL of several species, includ-ing humans, by the Nicotinamide adenine dinucleotidephosphate-diaphorase (NADPH-d) histochemistry tech-nique (Wassle et al., 1987; Huxlin, 1995). The threeisoforms of NOS are present in the retina (Goureau andCourtois, 1996). Using an antibody specifically directedagainst the C-terminal fragment of the neuronal NOSisoform, Perez et al. (1995) described NOS-ir neurons in

the GCL which could correspond to DACs and likely tosome GCs. Labeled neurons were also observed inside theINL near the inner border where 18-24% of Fos-ir cellswere double-labeled with NADPH-d (Koistinaho et al.,1993).

ACKNOWLEDGMENTS

The author thanks Dr. L. Martinet for her helpfulcomments on the manuscript, Dr. A. Saul and A. Bourochefor their critical reading and S. Venla for her technicalassistance. This work was supported by institutionalsupport of INRA.

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