activation of nmda receptors protects against glutamate neurotoxicity in the retina: evidence for...

Ž .Brain Research 827 1999 79–92

Research report

Activation of NMDA receptors protects against glutamate neurotoxicity inthe retina: evidence for the involvement of neurotrophins

Monica Rocha a,b,) , Rodrigo A.P. Martins a, Rafael Linden a

a Instituto de Biofısica Carlos Chagas Filho, UniÕersidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil´b Departmento de Farmacologia Basica e Clınica, Bl.J, Room J1-019, ICB, UniÕersidade Federal do Rio de Janeiro, Ilha do Fundao, 21941-590, Rio de´ ´ ˜

Janeiro, RJ, Brazil

Accepted 23 February 1999

Abstract

Activation of glutamate receptors has been implicated in excitotoxicity. Here, we have investigated whether subtoxic concentrations ofglutamate can modulate neuronal death in the developing retina. Explants of rat retinas were pre-incubated with glutamate, N-methyl-D-

Ž . Ž . Ž .aspartate NMDA , kainate, quisqualate or trans-1-amino-1,3-cyclopentanedicarboxylic acid t-ACPD for 18 h. Then, glutamate 6 mMwas added to the explants for an additional 6 h. Glutamate-induced degeneration was restricted to the emerging inner nuclear layer.Pre-incubation with glutamate, NMDA, or both, reduced glutamate-induced neuronal death and protected against neuronal death induced

Ž . Ž .by irradiation 2 Gy . The NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid d-APV; 30 mM or 5-methyl-10,11-dihydro-Ž .5H-dibenzocyclohepten-5,10-imine hydrogen maleate MK-801; 30 mM , prevented glutamate-induced neuroprotection. To investigate

whether this neuroprotection was mediated by neurotrophins, we incubated retinal explants with either brain-derived neurotrophic factoror neurotrophin-4. Both treatments resulted in partial protection against glutamate-induced neurotoxicity. Furthermore, NMDA mediatedneuroprotection was totally reversed when a soluble form of the specific tyrosine kinase receptor B was simultaneously added to theexplants. Our results suggest that activation of NMDA receptors may control neuronal death in the retina during development. Thismodulation seems to depend, at least in part, on the release of neurotrophins within the retina. q 1999 Published by Elsevier Science B.V.All rights reserved.

Keywords: N-Methyl-D-aspartate; Glutamate; Neurotoxicity; Apoptosis; Neuroprotection; Neurotrophins

1. Introductory statement

Glutamate is the major excitatory neurotransmitter inthe vertebrate retina. In the adult, glutamate is the transmit-ter for photoreceptors, bipolar cells, amacrine cells andganglion cells, mediating synaptic transmission in the pri-

w xmary visual pathway 2,10,11,49,60,75 . There are variouspharmacologically-defined glutamate receptor subtypes,most of which have been described in synapses within the

w xretina 33,54 . Besides its role as a neurotransmitter, gluta-mate is present early in fetal life, and participates inseveral developmental events, ranging from the shaping of

) Corresponding author. Departmento de Farmacologia Basica e´Clınica, Bl.J, Room J1-019, ICB, Universidade Federal do Rio de Janeiro,´Ilha do Fundao, 21941-590, Rio de Janeiro, RJ, Brazil. Fax: q55-21-˜590-1841 or q55-21-280-4694; E-mail: [email protected] [email protected]

dendritic architecture to the refinement of synaptic connec-w xtions 50,52 .

However, glutamate is also a potent excitotoxin toneuronal cells, and the administration of micromolar con-centrations of glutamate may lead to both immediate and

w xdelayed cell death 14,58 . Neuronal death involvingNMDA receptors, a glutamate receptor subtype activatedby N-methyl-D-aspartate, has been implicated in numerousacute and chronic neurodegenerative diseases, includingcerebral trauma, focal ischemia, epilepsy, Huntington dis-ease, Alzheimer disease, acquired immunodeficiency syn-

Ž .drome AIDS dementia and amyotrophic lateral sclerosisw x19 . In the retina, ischemic conditions such as retinalartery occlusion, diabetes mellitus, and some types ofglaucoma cause the release of an excessive amount of

w xglutamate, which results in retinal damage 22,53,80 . Incultured retinal neurons the activation of NMDA receptors,and subsequent influx of calcium, has been regarded as the

w xmain route for glutamate neurotoxicity 1,24,37,47 .

0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01307-4

( )M. Rocha et al.rBrain Research 827 1999 79–9280

Glutamate-induced cell death is generally characterizedby swelling of the cell soma, dendrites, and organelles,random DNA fragmentation, and lysis, which is associatedwith necrosis. However, it has been suggested that afterexcitotoxic injury, necrosis and apoptosis occur simultane-ously in the affected population, and it has been claimedthat internucleosomal DNA fragmentation, which is a hall-mark of apoptosis, is a common early step in either

w xnecrosis or apoptosis 64 . This molecular characterizationof apoptosis is also detected in cerebellar granule cellsfollowing glutamate administration, but several hours after

w xan early necrotic phase 3 . Moreover, internucleosomalDNA fragmentation also occurs in hippocampal neuronsfollowing ischemia and intracerebral injection of kainate or

w xglutamate 39,63,72 . However, other workers have failedto detect internucleosomal DNA fragmentation following

w xan excitotoxic insult 23,35,46 .During embryonic and postnatal development of the

nervous system, up to 85% of immature neurons areeliminated at the time they begin to establish synaptic

w xconnections with their targets 59 . Elimination of supernu-merary cell populations, which takes place during develop-ment, occurs mainly through apoptosis, and initial interac-tions of immature neurons with both their afferents and

w xtargets may determine neuronal survival 42 . In fact,neurotrophic factors released from target tissues protect

w xneurons from apoptosis during development 74 . How-ever, electric activity and neurotransmitters may have im-

w xportant roles as well 29,45 .Besides its well described role in neurotoxicity, gluta-

mate exerts an important neurotrophic activity upon sev-w xeral neuronal populations 6,7,15 . In fact, it has been

demonstrated that activation of glutamate receptors, espe-cially the NMDA subtype, and the influx of calciumthrough voltage sensitive calcium channels have an impor-tant role in the neuroprotection against deprivation of

w xneurotrophic factors 15,17,38,78 . There is increasing evi-dence that NMDA-mediated neuroprotection involves anincrease in mRNA to brain-derived neurotrophic factorŽ . Ž .BDNF and nerve growth factor NGF , and an increase

w xin protein synthesis 34,48 .In this study we investigated the effects of glutamate on

induced neuronal death in explants of the developingretina. We show that exposure to a subtoxic concentrationof glutamate andror NMDA can protect retinal neuronsfrom cell death induced by both glutamate and g-irradia-tion. We also tested whether the neuroprotective effect ofglutamate is a consequence of release of neurotrophins intothe retina.

2. Materials and methods

2.1. Preparation of explants

Lister hooded rats aging between the day of birth andŽ . Ž .postnatal day 4 P0–4 , and at postnatal day 30 P30 were

used. Experimental protocols were approved by the appro-priate institutional review committee. Explants of rat reti-

w xnae were obtained as previously described 4 . Briefly,when used at postnatal day 1, animals were instanta-neously killed by decapitation, and the eyeballs removed.P30 rats were anesthetized by ether inhalation, and thenkilled by decapitation. The retinae were dissected out andcut into small fragments of about 1 mm2, and each frag-ment considered one explant. Retinal explants obtainedfrom both eyes of 7 to 12 animals from the same litterwere pooled, and subsequently divided in various groupsaccording to the experimental protocol. Therefore, it isunlikely that explants under the same experimental condi-tion originated from the same eye, or the same animal.

ŽExplants were kept in basal medium Eagle BME, Gibco. ŽBRL supplemented with 5% fetal calf serum WL Im-

.munochemicals , sodium bicarbonate, glutamine and gen-tamicin, in an orbital shaker at 80–90 rpm, 5% CO and2

378C.Pharmacological agents were applied at the beginning

of the culture and kept for up to 24 h. Neurotoxic effectsof glutamate were examined by adding the neurotrans-mitter at 6 mM during the last 6 h of the culture. Whenglutamate antagonists were applied during pre-treatmentŽ .18 h of culture , explants were extensively washed withBME, and then exposed to 6 mM of glutamate for 6 h. In aset of experiments, explants were exposed to 2 Gy ofg-irradiation and examined after 3 h of culture.

2.2. Histology and data analysis

After 24 h of incubation, explants were fixed with 4%paraformaldehyde 0.1 M in phosphate buffer pH 7.4overnight, and kept in 20% sucrose for at least 12 h.Frozen 10 mm-thick transverse sections were cut, andmounted on gelatin-coated slides. The sections were thenstained with neutral red, and dehydrated with ethanol,cleared with xylene and coverslipped. Cells were analyzedby light microscopy, at 1000= magnification, and the rateof cell death in the retina was estimated from counts of

w xpyknotic nuclei, as in previous studies 65 .Each experiment consisted of testing distinct treatments

onto retinal explants obtained from the same pool asdescribed earlier. For each treatment, three to six differentretinal explants were analysed. From each explant, a single10 mm-thick section was selected, and pyknotic nuclei

Ž 2 .were counted in 3 distinct areas or fields 0.015 mmwithin the outer retinal stratum, and an average of thenumbers of pyknotic nuclei per field in each explant wasobtained. Data from distinct retinal explants were com-bined, and the mean was obtained for each particulartreatment. All results are expressed as mean"S.E.M. Thestatistical significance of the effects was calculated usinganalysis of variance followed by detailed comparisonsbetween groups using the Mann–Whitney procedure.

( )M. Rocha et al.rBrain Research 827 1999 79–92 81

2.3. Materials

Pharmacological agents glutamate, N-methyl-D-aspar-Ž .tate NMDA , kainate, quisqualate, 5-methyl-10,11-dihy-

dro-5H-dibenzocyclohepten-5,10-imine hydrogen maleateŽ . Ž .MK-801 , 2-amino-5-phosphonovaleric acid d-APV , D-

Ž .dinitroquinoxaline-2,3-dione DNQX , 6-cyano-7-nitro-Ž .quinoxaline-2,3-dione CNQX , a-methyl-4-carbo-

Ž .xyphenylglycine MCPG and trans-1-amino-1,3-cyclo-Ž .pentanedicarboxylic acid t-ACPD were obtained from

ŽResearch Biochemicals International RBI, Natick, MA,.USA . Neurotrophins BDNF and NT-4, and a soluble form

Žof the TrkB receptor tyrosine kinase neurotrophin receptor. ŽB conjugated with the Fc portion of human IgG TrkB-.IgG were provided by Regeneron Pharmaceuticals.

3. Results

Retinal explants from P1 rats were exposed to gluta-mate, at concentrations ranging from 1 mM to 30 mM, andobserved after 15 and 24 h of culture. Surprisingly, whenexplants were examined after 15 h of culture, only concen-trations of 10 mM or higher were able to induce degenera-

Ž .tion in the outer retinal stratum Fig. 1a . However, whenexplants were examined after 24 h, glutamate at 1 and 3mM induced an increase in the number of pyknotic nuclei

Ž .within the retina Fig. 1b . Therefore, when glutamate isadministered at the beginning of the culture, cells at theouter retinal stratum were resistant to 1 mM glutamate forup to 15 h of treatment, and at least 18 h of incubation are

Ž .needed for these developing cells to degenerate Fig. 1c .Interestingly, the peak of RGCs degeneration is also ob-served after 18 h of axotomy in vitro, suggesting thatfollowing RGC death, developing amacrine cells become

Ž .more sensitive to glutamatergic insult Fig. 1c, inset .Indeed, following 18 h of pre-incubation in controlmedium, 6 h of exposure to toxic concentrations of gluta-

Ž .mate 3 or 6 mM are enough to produce a significantŽ .increase in cell death within the retina Fig. 1d . In P1

retinal explants, after 18 h of pre-incubation in controlŽ .medium, glutamate 6 mMr6 h induced cell death in 90%Ž .of the experiments ns20 , leading to the finding of up to

a 40-fold increase in the number of degenerating cellsŽwhen compared with the control explants mean 14.27"

.2.5 . Moreover, when the retinae of rats aging between P2and P5 were examined, glutamate-induced cell death amongamacrine cells in the outer retinal stratum was observed in

Ž .all experiments ns5 , and the increase in the number ofdegenerating cells was more striking than in explants from

Ž .newborn rats 27.25"5.9; ns5 . At the end of the firstŽ .postnatal month P30 , retinal cells are very sensitive to

Ž .glutamate toxicity, and treatment with glutamate 6 mMfor the last 6 h in vitro resulted in 84"5 pyknotic nuclei

Ž .per field Fig. 1d .

An example of a typical experiment in which glutamateinduced cell death is shown in Fig. 2. In newborn rats, theouter stratum of the retina, which contains both the prolif-

Ž .erative zone, here termed the neuroblastic layer NBL anda few rows of early differentiating amacrine cells, is

Ž .separated from the ganglion cell layer GCL by a thinŽ .inner plexiforme layer ipl , which contains neuronal pro-

Ž .cesses and migrating displaced amacrine cells Fig. 2a .Retinal explants kept in vitro maintain the histotypic orga-

w xnization of the retina of the same age in situ 43,65 .However, 24 h after sectioning the optic nerve, most

Ž .retinal ganglion cells RGCs have already degenerated.Glutamate-induced neuronal death is characterized by

condensation and fragmentation of chromatin, and wasmostly observed within the innermost portion of outer

Ž .retinal stratum Fig. 3a . This region is occupied byamacrine cells and represents the emerging inner portion

Ž .of the inner nuclear layer INLi . The localized cell deathprobably reflects the expression of glutamate receptors indeveloping cells, and suggests that differentiated cells aremore sensitive to glutamate than the proliferating andundifferentiated cells in the NBL. According to this as-sumption, RGCs, which are the first to become postmi-totic, should be more sensitive to glutamate than amacrinecells. Indeed, while cells at the outer retinal stratum wereresistant to 1 mM glutamate for up to 15 h of treatment,RGCs degeneration was accelerated during the first 4 h ofglutamate incubation, resulting in an increase in the per-centage of pyknotic nuclei from 2.21"0.97 to 16.58"

Ž1.56, in the control and treated groups, respectively data.not shown . As discussed earlier, at P30, neuronal death

induced by glutamate was also greater than that observedat P0–1, and pyknotic nuclei were observed throughout the

Ž .inner and outer nuclear layers Fig. 3d, inset . Theseresults probably reflect the fact that, by P30, most retinalcells are differentiated and express glutamate receptors.

To investigate which subtypes of glutamate receptorsare involved in the induced neuronal death, explants were

Žexposed during the last 6 h of incubation to glutamate 6.mM in the presence of either one of the NMDA receptor

Ž . Ž .antagonists d-APV 100 mM or MK-801 100 mM , orŽwith the non-NMDA receptors antagonist DNQX 100

.mM . Glutamate-induced neuronal death was preventedŽwith either d-APV or MK-801, but not with DNQX Fig.

.3b . Therefore, induced cell death observed in the emerg-ing INL is due to specific activation of the NMDA subtypeof glutamate receptors.

Administration of a subtoxic concentration of glutamateŽ .60 mM during the first 18 h of culture induced areduction of 41% to 67% of glutamate-induced cell deathŽ .mean 59.01"4.66% . In retinal explants from P1 rats,

Ž .this effect was observed in all experiments ns9 , anexample of which is shown in Fig. 3c. In retinal explantsfrom rats aging P2–4, this reduction in neurotoxicity was

Ž .somewhat smaller 20 to 38%; mean 28.05"5.42; ns3 .The glutamatergic agonist NMDA was also tested and


Ž .Fig. 2. Protective effect of glutamate on glutamate-induced neurotoxicity. Photomicrographs of transverse sections through explants of P1 rat retina. AŽ . Ž .Control explant after 24 h in vitro, showing cells the neuroblastic layer NBL and emerging amacrine cell layer INLi separated from cells at the ganglion

Ž . Ž .cell layer by a thin inner plexiforme layer. RGCs degenerate after sectioning the optic nerve asterisk . B Explant maintained in control medium for 18 h,Ž . Ž . Ž .and kept for additional 6 h in vitro with glutamate 6 mM , showing pyknotic nuclei within the emerging amacrine cell layer arrow . C Explant

Ž .pre-treated with glutamate 60 mM for 18 h, and kept for the last 6 h with glutamate 6 mM is protected from degeneration within the outer retinal stratum.INLi: inner portion of the inner nuclear layer. Calibration bar: 20 mm.

showed a similar rate of protection than that observed withŽ . Žglutamate Fig. 3c,d . Neuroprotection with NMDA 60

. Ž .mM was observed in 92% of the experiments ns13 ,and there was a reduction of 30% to 96.2% in the number

Ž .of pyknotic nuclei mean 58.16"5.05 . In one experi-ment, both glutamate and NMDA were added simultane-ously during the pre-treatment, and maximal neuroprotec-

Ž .tion was observed Fig. 3c . Explants were treated withconcentrations of NMDA ranging from 1 mM to 300 mMfor 24 h, and none of these tested concentrations induced

Ž .any degeneration in retinal explants data not shown .Therefore, 60 mM was considered safe and subtoxic forthe pre-treatment. Glutamate and NMDA-induced protec-tion was also observed at P30, where the reduction in

Ž .glutamate-induced cell death was around 70% Fig. 3d .

To further examine which receptor subtype mediatesglutamate-induced protection against glutamate neurotoxic-ity, selective antagonists and agonists of either NMDA ornon-NMDA receptors were used. Antagonists were appliedsimultaneously with glutamate during the pre-treatment,and extensively washed out before administration of neuro-

Ž .toxic glutamate 6 mM . The NMDA receptor antagonistMK-801 was tested in concentrations of 3, 10 and 30 mMŽ .ns5 . These concentrations totally or partially reversedglutamate-induced neuroprotection, with the highest con-centration being the most effective. To further examine theparticipation of NMDA receptors in the neuroprotectioninduced by glutamate, we tested d-APV, in the concentra-tions of 6, 30 and 100 mM, and neuroprotection wastotally blocked by all concentrations in all experiments

Fig. 1. Dose response and time course of glutamate toxicity in retinal explants. Results are expressed as the number of pyknotic nuclei per field in the outerŽ . Ž .retinal stratum. A Explants of P1 rat retina were exposed to various concentrations of glutamate ranging from 30 mM to 30 mM for 15 h. A significant

Ž .effect was observed with 10 mM of glutamate. B Explants of P1 rat retina were exposed to glutamate at concentrations ranging from 1 mM to 3 mM, andobserved after 24 h in vitro. After this longer period of incubation, glutamate at 1 and 3 mM induced an increase in the number of pyknotic nuclei within

Ž . Ž .the retina. C Glutamate 1 mM was applied at the beginning of the culture and kept for up to 24 h. At least 18 h of incubation were required forsignificant cell death, a period that corresponds to the peak of RGC degeneration after axotomy. Inset: time course of ganglion cell death in retinal explants

Ž . Ž .after axotomy, shown as the percentage of pyknotic nuclei in the ganglion cell layer GCL . D Glutamate toxicity in retinal explants increases withdevelopment. Retinal explants from rats at P1, P3, P5 or P30 were incubated in control medium for 18 h, and then treated with 6 mM of glutamate for anadditional 6 h. Each symbol and bar indicates the mean and S.E.M., respectively.


Ž .Fig. 3. Glutamate-induced neurotoxicity and neuroprotection depend on NMDA receptor activation. A Glutamate-induced cell death is restricted to theŽ .innermost third of the neuroblastic layer of explants of P1 rat retinae, where more than 80% of the pyknotic nuclei were located. B Glutamate-induced

Ž . Ž . Ž . Ž .neurotoxicity is blocked by d-APV 100 mM and MK-801 100 mM , but not following simultaneous application of DNQX 100 mM . C NeurotoxicityŽ . Ž . Ž .is partially blocked by pre-treatment with glutamate 60 mM , NMDA 60 mM or both. D Glutamate-induced protection was also observed at P30, when

Ž . Ž .neurotoxicity was distributed through the entire outer retina inset . The ordinate indicates the number or percentage, in A of pyknotic cells per field inthe outer retinal stratum, and each column and bar indicates the mean and S.E.M. U p-0.05, UU p-0.01, UUU p-0.001, significantly different fromglutamate-treated groups.

Ž . Ž .ns2 Fig. 4a . d-APV alone in concentrations up to 100mM is not toxic to retinal explants treated for up to 24 hŽ .data not shown . Similarly, MK-801 was applied to retinalexplants in concentration ranging from 0.3 to 100 mMduring 24 h of culture, and none of these concentrations

Ž .were neurotoxic data not shown . On the other hand, theŽ .non-NMDA receptor antagonists DNQX 30 mM and

Ž .CNQX 30 mM were always ineffective in blocking gluta-

Ž .mate-induced neuroprotection ns4 . DNQX was appliedalone to retinal explants for 24 h in concentrations rangingfrom 0.3 to 100 mM, and did not induce degeneration in

Ž . Ž .the neuroblastic layer data not shown . Kainate 60 mMŽ .and quisqualate 60 mM , specific non-NMDA receptor

agonists, were also tested, and no neuroprotection wasobserved following pre-incubation with either of theseagents. Participation of metabotropic glutamate receptors

Ž . Ž . Ž .Fig. 4. Protective effect of glutamate is blocked by NMDA antagonists. A When explants were incubated with d-APV 30 mM or MK-801 30 mMŽ . Ž .during the first 18 h, glutamate neuroprotection was prevented, while application of DNQX 30 mM was ineffective. B Neuroprotection was observed

Ž . Ž . Ž . Ž .after chronic administration of NMDA 60 mM , but not kainate 60 mM , quisqualate 60 mM or t-ACPD 300 mM . The vertical axis indicates thenumber of pyknotic nuclei per field in outer retinal stratum, and each column and bar indicates the mean and S.E.M.


Fig. 5. Protective effect of glutamate on g-irradiation-induced neurotoxicity. Photomicrographs of transverse sections through explants of P1 rat retina,Ž . Ž .showing glutamatergic protection against cell death induced by g-irradiation 2 Gy . A Control explant after 21 h in vitro. The borderline between the

Ž .neuroblastic layer and the inner portion of the inner nuclear layer is indicated by the dashed line. B Explant kept in control medium for 18 h, g-irradiated,Ž . Žand further maintained for an additional 3 h in vitro, showing extensive cell death in the neuroblastic layer arrow and emerging amacrine cell layer arrow

. Ž . Ž .with asterisk . C Explants pre-treated with glutamate 60 mM for 18 h, and then g-irradiated, showing protection from degeneration only in theinnermost portion of the outer retinal stratum. Calibration bar: 20 mm.

was also tested, but neither their specific agonist t-ACPDŽ . Ž .300 mM nor the antagonist MCPG 300 mM was effec-tive in either inducing or blocking neuroprotection, respec-

Ž . Ž . Ž .tively ns4 Fig. 4 . t-ACPD 30 to 300 mM adminis-tered to retinal explants during 24 h had no effect on cellviability. We have also failed to protect retinal cells fromglutamate excitotoxicity by pre-incubating the explantsunder a general depolarization condition provided by 50

Ž .mM potassium data not shown . Therefore, whereasNMDA significantly protected retinal neurons against glu-tamate-induced neuronal death, none of the other gluta-

Ž .matergic agonists tested had protective effects Fig. 4b .Moreover, none of the glutamate agonists and antagonistsat the concentrations used in this study affected neuronalviability when applied alone to retinal explants for up to24 h. Therefore, the neuroprotective effect of glutamateseems to depend on NMDA receptor activation.

Chronic treatment of retinal explants with glutamateŽ .also prevented cell death induced by g-irradiation Fig. 5 .

ŽWhen retinal explants were exposed to g-irradiation 2.Gy after pre-incubation for 18 h in control medium,

Ž .extensive neuronal death was observed Fig. 5b . Pre-treat-Ž .ment with subtoxic concentration of glutamate 60 mM

partially protected neurons against irradiation-induced neu-Ž .ronal death Fig. 5c, Fig. 6a . To investigate if cell death

induced by irradiation results from increased amount ofglutamate within the retina, we treated explants with d-APVŽ .100 mM both during and after the insult. Fig. 6b showsthat degeneration is not affected by simultaneous adminis-tration of the NMDA receptor antagonist, d-APV. More-over, there were some differences between g-irradiationand glutamate-induced cell death. First, cell death wasmore extensive after irradiation, and up to 20 times morepyknotic nuclei were observed. Furthermore, irradiation-

Ž . ŽFig. 6. Neuronal death induced by g-irradiation and its prevention by glutamate. A After 18 h in vitro, retinal explants were exposed to g-irradiation 2. Ž .Gy . After 3 h of incubation, extensive neuronal death is observed black column . Neuronal death was partially prevented by pre-treatment of explants

Ž . Ž . Ž .with 60 mM glutamate dotted column . B Neuronal death induced by g-irradiation was not affected by administration of d-APV 100 mM both duringŽ . Ž .and after exposure of explants to irradiation. C After irradiation black columns , degenerating cells were found throughout the outer retinal stratum, but

Ž .were preferentially located in outer portions of the neuroblastic layer. Pre-treatment with 60 mM glutamate dotted columns prevented neuronal death inŽ .the innermost and intermediate portions of the neuroblastic layer. This neuroprotective effect was blocked when d-APV 100 mM was applied together

Ž .with glutamate during 18 h of incubation hatched columns . The vertical axis indicates the number of pyknotic nuclei per field in the neuroblastic layer,and each column and bar indicates the mean and S.E.M.


induced cell death was extended to outer portions of theNBL, where pyknotic nuclei were preferentially locatedŽ .Fig. 6c . However, glutamate-induced neuroprotectionagainst irradiation was restricted to the inner and middleportions of the outer retinal stratum, thus favoring differen-tiated amacrine cells. Neuroprotection against irradiationwas again blocked when d-APV was simultaneously addedwith glutamate during the pre-treatment, showing its de-pendence on the activation of NMDA receptors.

To investigate both the effects of neurotrophins andtheir possible involvement in the neuroprotection mediatedby NMDA receptors, retinal explants were pre-incubated

Ž . Ž .with either BDNF 30 ngrml or NT-4 10 ngrml . Bothneurotrophins protected explants against glutamate-in-duced neuronal death in all experiments in which NMDAalso showed a neuroprotective effect. Neuroprotection withBDNF was very similar to the one observed with NMDA,and ranged from 22.5 to 74% of reduction in glutamate

Ž . Ž .toxicity mean 48.05"10.83; ns4 Fig. 7a . Retinal

explants were also treated with NT-4, which also binds toTrkB neurotrophin receptors. The neuroprotective effectobserved with NT-4 was more pronounced than that withNMDA, ranging from 65.8% to 91.4% of reduction in

Ž .glutamate-induced cell death mean 80.26"7.6; ns3Ž .Fig. 7b . Therefore, both neurotrophins mimic the neuro-protective effect of NMDA against glutamate-induced cell

Ž .death. BDNF 1–300 ngrml , when applied alone toretinal explants for 24 h, had no effect on neuronal viabil-

Ž . Žity in all experiments ns3 . However, NT-4 1–30.ngrml , when administered alone reduced cell death by

86.4% at the outer retinal stratum in one experiment, withŽ .no effect in the other 2 experiments ns3 .

To examine whether chronic treatment with either glu-tamate or NMDA may protect retinal cells through therelease of neurotrophins, explants were treated with a

Žsoluble form of the TrkB neurotrophin receptor TrkB-IgG;. Ž .300–900 ngrml . The ‘receptor body’ TrkB-IgG at the

concentration of 900 ngrml completely prevented

Ž . Ž .Fig. 7. Glutamate-induced neurotoxicity is prevented by neurotrophins. A Explants kept for 18 h with BDNF 30 ngrml were protected fromŽ . Ž . Ž .glutamate-induced neuronal death similarly to explants chronically pre-treated with NMDA 60 mM . B Pre-treatment of explants with NT-4 10 ngrml

Ž .also mimicked NMDA-induced protection. C NMDA-induced neuroprotection was antagonized by TrkB-IgG in a dose-dependent fashion, and while theconcentration of 300 ngrml partially prevented neuroprotection, 900 ngrml completely antagonized it. Administration of TrkB-IgG alone had no effect oncell viability. The vertical axis indicates the number of pyknotic nuclei per field in the outer retinal stratum, and each column and bar indicates the meanand S.E.M. U p-0.05, UU p-0.01, UUU p-0.001, significantly different from glutamate-treated groups.


NMDA-induced protection against glutamate neurotoxicityŽ . Ž .in all experiments ns3 Fig. 7c . Administration of

TrkB-IgG for 24 h alone did not affect cell viability in theŽ .outer retinal stratum ns3 .

4. Discussion

In this study we have shown that retinal cells located inthe emerging inner nuclear layer of the retina of newbornrats die when retinal explants are exposed to high concen-trations of glutamate. However, when glutamate is admin-istered at the beginning of the culture, at least 18 h ofincubation are needed for these developing cells to degen-erate. On the other hand, following 18 h of pre-incubationin control medium, 6 h of exposure to toxic concentrationsof glutamate are enough to produce cell death. Likewise,the peak of RGCs degeneration is also observed after 18 hof axotomy in vitro, suggesting that following RGC death,developing amacrine cells become more sensitive to gluta-matergic insult. Therefore, it is possible that interactionsbetween RGCs and cells at the emerging inner nuclearlayer affect neuronal sensitivity to glutamatergic excitotox-icity during development of the retina.

In the present study, 6 mM of glutamate was required toinjure cells at the emerging inner nuclear layer of retinalexplants of P1 rats. The resistance of developing retinalcells to glutamate excitotoxicity has been shown else-where, where contrary to expectations, glutamate up to 5mM greatly increased the survival of dissociated neonatalretinal ganglion cells, with a possible toxic effect observed

w xonly at concentrations of 10 mM 57 . Glutamate-inducedcell death involves the activation of NMDA receptors, asshown by the effects of specific antagonists. The involve-ment of NMDA receptors in glutamate-induced cell deathin the rat retina has been reported elsewhere, and amongretinal neurons, amacrine cells seem to be the most sensi-

w xtive to NMDA neurotoxic insult 1,32,37,70 . The locationof the sensitive cells in our experiments is consistent withtheir identification as early developing amacrine cells, andthey can be stained with antibodies to calretinin, which

Ž .labels amacrine cells unpublished data . Moreover, thelocation of these cells is consistent with the area in whichnitric oxide synthase immunoreactivity and positiveNADPH-diaphorase cells are observed in the immature

w xretina 57,77 . Indeed, it has been suggested that therelease of nitric oxide by retinal cells may be one of themechanisms mediating glutamate-induced neuronal death

w xin the neonatal rat retina 57 . We are currently investigat-ing the involvement of nitric oxide in both the neurodegen-eration and neuroprotection induced by glutamate in retinalexplants.

Besides its well known effects on dendritic pruning andneurite sprouting, it is likely that glutamate plays animportant role in the control of cell death in the developingretina. At birth, prior to differentiation of many neuronalcell types and before the onset of synaptogenesis, gluta-

mate is found diffusely distributed across the rat retinaw x26 . Moreover, during early postnatal development of theretina, there is a high concentration of extracellular gluta-mate, which is reduced to considerably lower levels in the

w xadult 31 . High concentrations of glutamate might be aconsequence of the inability of immature Muller and other¨retinal cells to remove glutamate from the extracellularspace. Glutamate is neurotoxic to cortical immature neu-rons even before the development of glutamate receptor-mediated currents, through a mechanism that probablyinvolves competitive inhibition of high affinity cystineuptake, resulting in glutathione depletion and accumulation

w xof cellular oxidants 55 . However, since NMDA is notable to inhibit cystine uptake, this mechanism is not re-sponsible for the glutamate excitotoxicity in the retina inour studies. Moreover, it has been shown that glutamatedoes evoke depolarizing currents in nearly all RGCs in P5rat retinae, within the period of naturally occurring gan-

w xglion cell death 62,67 . Therefore, it is possible thatfunctional glutamatergic input participates in histogeneticcell death in the retina.

Our results also show that chronic treatment with eitherglutamate or NMDA in micromolar concentrations protectsretinal cells against glutamate-induced cell death. Thiseffect was prevented by either d-APV or MK-801, whileantagonists of non-NMDA receptors were ineffective. Thedata suggest that NMDA receptors mediate neuroprotec-tion against glutamate-induced cell death. In the retina, itis well described that electrical activity is important forRGCs survival during the period of naturally occurring celldeath, while glutamate prevents neuronal death and en-

w xhances the sprouting of neurites in RGCs 45,57 . More-over, chronic depolarization prevents apoptosis induced by

w xtrophic factor deprivation in several neuronal types 27,28 .Both the activation of glutamate receptors, especially theNMDA receptor subtype, and calcium influx through volt-age-gated calcium channels participate in the mechanism

w xof neuroprotection 15,17,18,28,38 . Therefore, glutamatemay have both toxic and trophic effects during neuronal

w xdevelopment 50,51 .Reduced glutamate toxicity after chronic treatment with

NMDA could be a consequence of receptor desensitizationor down-regulation. However, we have shown that gluta-mate also prevents cell death induced by g-irradiation. Themechanism by which irradiation induces degeneration is

w xnot fully understood 56 , but we have shown that it doesnot involve NMDA receptor activation. Moreover, gluta-mate or NMDA-induced neuroprotection was also ob-served when these agents were completely washed outafter the pre-treatment, suggesting that their continuouspresence is not required for neuronal protection to occur.In conclusion, it is unlikely that glutamate-induced neuro-protection results from receptor desensitization.

NMDA receptors constitute the major pathway respon-sible for transcriptional regulation induced by glutamatew x5,16,40 . Calcium entry through NMDA receptors induces


gene expression via activation of the Ras–Raf-mitogen-Ž . w xactivated protein kinases MAPKs signaling cascade 68 .

In rat cortical neurons, phosphorylated MAPK translocatesto the nucleus, where it induces gene expression via a

Ž . w xternary complex factor TCF linked pathway 76 . Inhippocampal neurons, calcium signals can directly stimu-late transcription via serum response factor binding site,through both Ras–MAP kinase and TCF-independent

w xmechanisms 36 . Therefore, there are several pathways bywhich glutamate and calcium control gene expression andregulate neuronal survival.

Our findings show that the neurotrophins BDNF andNT-4 protect retinal cells against glutamate-induced celldeath. In neonatal rat retinae, the expression of mRNAcoding for BDNF and trkB receptor is restricted to theganglion cell layer and proximal, more-differentiated, neu-

w xroblastic layer 61,66 . Similarly to glutamate-induced geneexpression, activation of TrkB receptors by BDNF or NT-4also leads to activation of Ras–Raf-MAP kinase signalling

w xcascade, interfering with the survival of target cells 71 .Although Ras constitutes an important intracellular path-way for neurotrophin effects, several Ras-independent

w xpathways have been described as well 21,79 . Moreover, ithas been shown that BDNF enhances intracellular calciumconcentration in hippocampal neurons through a mecha-nism that probably involves generation of inositol trisphos-phate, and consequent release of calcium from the endo-

w xplasmic reticulum into the cytoplasm 8,9 . So, it is possi-ble that intracellular calcium increase through this pathwaycould underly a mechanism for neuroprotection similar tothat induced by NMDA receptor activation. Indeed, pre-treatment of hippocampal neurons with BDNF protects

w xagainst excitotoxic insults 13 . Therefore, both glutamateand neurotrophins are involved in the regulation of cal-cium influx into developing neurons, which in turn, canactivate several pathways for regulating neuronal survival.Ras and Ras-related proteins, which can be activated byboth calcium and neurotrophins, have been shown to mod-ulate ion channel expression, cytoskeletal proteins, andother second messenger systems that interfere with both

w xneuronal excitability and neurotoxicity 25,41,69 .In the retina, the neuroprotective effect of glutamate

seems to depend on the release of BDNF andror NT-4,since it was prevented when explants were exposed to asoluble form of TrkB receptor. The fact that neuroprotec-tion could be completely eliminated when TrkB-IgG wasadded to the media during the pre-treatment suggests thatno other receptor types for neurotrophins are needed forthis effect. Therefore, calcium entry through NMDA recep-tors may lead to secretion of BDNF andror NT-4, andactivation of TrkB receptors, which results in neuroprotec-tion. Indeed, previous studies have shown that NMDA-mediated neuroprotection requires new RNA and proteinsynthesis, and that glutamate induces expression of BDNF

w xand NGF mRNAs 20,34,48,81 . Also, it has been shownthat administration of anti-BDNF antibodies to cortical

neurons blocks depolarization-induced, Ca2q-dependent,w xneuronal survival 30 . All these data are consistent with

the sequence of events described above. However, toconfirm this hypothesis, we are currently investigating ifNMDA receptor activation increases neurotrophin expres-sion.

Moreover, we cannot exclude the possibility that addi-tional neurotrophic factors also play a role in the neuropro-tection against glutamate-induced cell death within theretina. Indeed, it has been shown that NT-3, BDNF, NGF,epidermal growth factor, basic fibroblast growth factor andastrocyte conditioned medium protect various neuronal

w xtypes against glutamate toxicity 12,13,44,73 . These re-sults are consistent with the idea that various growthfactors regulate neuronal survival during development.

In conclusion, our findings show that glutamate func-tions as a neuroprotective agent against retinal cell death.During development of the retina, neurons may requireboth neuronal activity through NMDA receptors and ade-quate amounts of neurotrophic factors, whenever chal-lenged by potentially lethal insults.

Acknowledgements

The authors express their thanks to Helena Borges andMargareth Nakatani for their support, to Jose Nilson dosSantos for technical assistance, and Regeneron Pharmaceu-ticals for kindly providing the neurotrophins. This workwas supported by CNPq, FAPERJ, FINEP and PRONEX-MCT-1072.

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