metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure

Metabotropic glutamate receptors are differentially regulated under

elevated intraocular pressure

Frank M. Dyka,* Christian A. May� and Ralf Enz*

*Institut fur Biochemie, Emil-Fischer-Zentrum and �Anatomisches Institut II, Friedrich-Alexander-Universitat Erlangen-Nurnberg,

Erlangen, Germany

Abtract

Glaucoma is a leading cause of blindness, ultimatively

resulting in the apoptotic death of retinal ganglion cells.

However, molecular mechanisms involved in ganglion cell

death are poorly understood. While the involvement of iono-

tropic glutamate receptors has been extensively studied, vir-

tually nothing is known about its metabotropic counterparts.

Here, we compared the retinal gene expression of metabo-

tropic glutamate receptors (mGluR) in eyes with normal and

elevated intraocular pressure (IOP) of DBA/2J mice, a model

for secondary angle-closure glaucoma using RT–PCR and

immunohistochemistry. Elevated IOP in DBA/2J mice signifi-

cantly increased retinal gene expression of mGluR1a,

mGluR2, mGluR4a, mGluR4b, mGluR6 and mGluR7a when

compared to C57BL/6 control animals, while mGluR5a/b and

mGluR8a were decreased and no difference was observed for

mGluR3 and mGluR8b. Specific antibodies detected an

increase of mGluR1a and mGluR5a/b in both synaptic layers

and in the ganglion cell layer of the retina under elevated IOP.

Because ganglion cell death in DBA/2J mice occurs most

likely by apoptotic mechanisms, we demonstrated up-regula-

tion of mGluRs in neurons undergoing apoptosis. In summary,

we support the idea that the specific gene regulation of

mGluRs is a part of the glaucoma-like pathological process

that develops in the eyes of DBA/2J mice.

Keywords: apoptosis, gene expression, glaucoma, immun-

ocytochemistry, retina, RT–PCR.

J. Neurochem. (2004) 90, 190–202.

Glaucoma is a prevalent cause of blindness, ultimatively

resulting in the apoptotic death of retinal ganglion cells. In

recent years, several hypothesis have been formulated that

could explain the molecular mechanisms that cause injury

and death of ganglion cells. Besides an often observed

increased intraocular pressure (IOP), described factors

include genetic predisposition (Stone et al. 1997; Fingert

et al. 1999; Stoilov et al. 2002; Vincent et al. 2002), nitric

oxide (Liu and Neufeld 2001) and c-synuclein (Surgucheva

et al. 2002), reduced availability of nerve growth factors

(Pease et al. 2000; Quigley et al. 2000), the involvement of

receptors for the excitatory neurotransmitter glutamate

(Kreutz et al. 1998; Lam et al. 1999; Kwong and Lam

2000), of heat-shock proteins (Tezel et al. 2000; Park et al.

2001; Salvador-Silva et al. 2001) and of antibodies directed

against retinal proteins (Ikeda et al. 2000; Schori et al.

2001). However, detailed molecular mechanisms involved in

ganglion cell death are still poorly understood.

In the mammalian CNS, glutamate mediates excitatory

neurotransmission via ion channel-associated (ionotropic)

and G protein-coupled (metabotropic) receptors. While

ionotropic NMDA-, AMPA- and kainate-type glutamate

receptors mediate fast synaptic transmission, metabotropic

glutamate receptors (mGluRs) modulate neuronal excitability

and development, synaptic plasticity, transmitter release and

memory function using a variety of intracellular second

messenger systems (Hollmann and Heinemann 1994; Nak-

anishi et al. 1998). To date, eight different members of the

mGluR family have been cloned, which are subdivided into

three groups, based on sequence homology, associated

second messenger systems and pharmacological properties

(Pin and Duvoisin 1995; Conn and Pin 1997).

Received November 18, 2003; revised manuscript received February 19,

2004; accepted February 23, 2004.

Address correspondence and reprint requests to Dr Ralf Enz, Institut

fur Biochemie, Friedrich-Alexander-Universitat Erlangen-Nurnberg,

Fahrstr.17, D-91054 Erlangen, Germany.

E-mail: [email protected]

Abbreviations used: CT, cycle of threshold; EF1a, elongation factor

1a; FCS, fetal calf serum; GABACR, c-aminobutyric acid type C

receptor; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner

plexiform layer; IOP, intraocular pressure; mGluR, metabotropic glu-

tamate receptor; ONL, outer nuclear layer; OPL, outer plexiform layer;

PBS, phosphate-buffered saline.

Journal of Neurochemistry, 2004, 90, 190–202 doi:10.1111/j.1471-4159.2004.02474.x

190 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 190–202

In the retina, abnormal elevated levels of glutamate can

perturb the neuronal glutamatergic system and finally result

in the death of ganglion cells (Vorwerk et al. 1996; Otori

et al. 1998). The NMDA-type glutamate receptors have been

shown to play a major role in apoptotic ganglion cell death

(Lam et al. 1999; Kwong and Lam 2000), and their

expression was altered in excitotoxic disease states (Samar-

asinghe et al. 1996; Kreutz et al. 1998; Lam et al. 1999;

Kwong and Lam 2000). Although the original observation of

elevated glutamate concentrations in the vitreous of glau-

coma patients could not be verified (Dreyer et al. 1996;

Carter-Dawson et al. 2002), glutamate might still play a

major role in the disease (Carter-Dawson et al. 2002). As

glutamate receptors are clustered to a large extend at synaptic

specializations (Dev et al. 2001; Sheng 2001), the local

concentration of the neurotransmitter at synapses seems to be

far more important for its role in excitotoxicity than the

average glutamate concentration of the vitreous.

Whereas the involvement of ionotropic glutamate recep-

tors, especially of the NMDA-type, in the pathogenesis of

chronic glaucoma was extensively studied, virtually nothing

is known about the relation between metabotropic glutamate

receptors and retinal degeneration. In this study, we inves-

tigated the expression of metabotropic glutamate receptors at

increased IOP, using a mouse model for secondary angle-

closure glaucoma (DBA/2J; John et al. 1998) and a rat

retinal ganglion cell line (Krishnamoorthy et al. 2001).

Materials and methods

IOP measurement and immunocytochemistry

IOP was measured in the eyes of 2- and 6-month old DBA/2J mice

using a microneedle system as described in the literature (John et al.

1997). The animals were deeply anaesthetized with an overdose of

thiopental and placed on a surgical platform. The eye was viewed

under a dissecting microscope, and the microneedle (size 30 G) tip

was placed inside a drop of phosphate-buffered saline (PBS) on top

of the eye. At this point, the pressure reading was zeroed. The tip of

the microneedle was manually inserted into the anterior chamber

with the IOP recorded continuously. The IOP values usually reached

a plateau after 1 s of higher IOP values due to the needle insertion. If

the plateau was constant for 1 min, the eye was given some gentle

extra pressure from outside to confirm microneedle patency. This

manipulation resulted in some IOP peaks that returned to the prior

plateau level. The microneedle was then withdrawn from the

anterior chamber; rapid return of the pressure to zero was required

for the inclusion of data. The original measurements were calibrated

in cmH2O, and then converted into mmHg for comparison with the

literature.

Eyes of 2- and 6-month-old DBA/2J mice, where IOP was

recorded prior to enucleation, were fixed in 4% paraformaldehyde

for 4 h and then washed in PBS. Sagittal frozen 12-lm-thick

sections were cut with a cryotome, mounted on glass slides and

incubated with Blotto’s dry milk solution for 20 min at room

temperature to reduce non-specific background staining. Incubation

of the sections was performed overnight at 4�C with the primary

antibodies recognizing mGluR1a (diluted 1 : 500; Sigma, Taufkir-

chen, Germany) or with both mGluR5 splice variants (diluted

1 : 200; Upstate Biotechnology, Lake Placid, NY, USA) in PBS

containing 1% bovine serum albumin and 0.3% Triton X-100.

Sections were rinsed in PBS three times and incubated with a

secondary Cy3-conjugated antibody (1 : 2000; Dianova, Hamburg,

Germany) for 1 h at room temperature. After another rinsing the

sections were mounted with Kaisers glycerine jelly (Merck,

Darmstadt, Germany) and viewed under a Leica fluorescence

microscope (Leica, Bensheim, Germany). Only retinal pieces from

the same eccentricity were compared. Control sections were

performed as described but omitting the primary antibody.

RNA purification from mouse retinae

Total RNAwas extracted from retinae of 2- and 6-month-old DBA/2J

and C57BL/6 mice following a method described by Chomczynski

and Sacchi (1987). To avoid changes in gene regulation between

individuals, retinae from two to five animals were pooled. Briefly,

retinal tissues were disrupted in 0.3 mL denaturation buffer [4 M

guanidinium thiocyanate, 0.7% b-mercaptoethanol (v/v), 25 mM

sodium citrate, 0.5% sodium-lauroyl-sarkosin (w/v); Sigma, St

Louis, MO, USA] using glass beads. After adding 2 M sodium

acetate (pH 4.0), acid phenol and chloroform/isoamylalcohol

(24 : 1), RNA extraction and sedimentation was performed accord-

ing to the protocol of Chomczynski and Sacchi (1987). To remove

possible contamination from chromosomal DNA, the precipitated

RNA was incubated for 30 min at 37�C with 50 Units DNaseI

(Roche Diagnostics, Mannheim, Germany) and 40 Units RNasin

(Roche Diagnostics) in a final volume of 20–40 lL, followed by

acid phenol extraction and ethanol precipitation. The RNA

concentration was measured photometricaly and the RNA was

stored at )80�C.

CDNA synthesis

One microgram of total RNA of each retina type was reverse-

transcribed using the ‘Smart cDNA synthesis kit’ (Clontech, Palo

Alto, CA, USA) according to the manufacturer’s protocol. Briefly,

total RNA was mixed with 1 lL modified oligo(dT) CDS primer

(10 lM), 1 lL SMART II oligonucleotide (10 lM) and DEPC

treated water to a total volume of 5 lL. After heating the mixture at

70�C for 2 min, 2 lL 5 · buffer (250 mM Tris–HCl pH 8.3,

375 mM KCl, 30 mM MgCl2), 1 lL dithiotreitol (20 mM), 1 lLdNTPs (10 mM each) and 1 lL Powerscript II reverse transcriptase

were added. The reaction mix was incubated at 42�C for 2 h and

diluted with 40 lL TE (10 mM Tris–HCl pH 8.0, 1 mM EDTA).

Subsequently, 1 lL of the diluted sample was used for cDNA-

amplification. The optimum number of amplification cycles was

determined as described in the protocol. Amplified cDNA was

purified using the concert rapid PCR purification system (Gibco

BRL, Grand Island, NY, USA), the concentration of the obtained

cDNA was measured photometricaly and adjusted to 10 ng/lL for

all retina samples.

PCR amplification and data analysis

Semi-quantitative RT–PCR was performed using 10 ng cDNA as a

template in 20 lL of PCR buffer (20 mM Tris–HCl pH 8.0, 50 mM

KCl, 2.5 mM MgCl2, 0.2 mM dNTPs, 2.5 mM each primer, see

mGluR expression in the retina of DBA/2J mice 191

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 190–202

Table 1), 1 Unit Taq-polymerase; Invitrogen, Karlsruhe, Germany)

in a programmable thermocycler (GeneAmp PCR System 9700;

Applied Biosystems, Foster City, CA, USA) using the following

parameters: 94�C for 3 min followed by 20 or 25 cycles at 94�C for

30 s, 62�C for 45 s, 72�C for 45 s and a final incubation at 72�C for

10min. Each RT–PCR experiment was performed at least three

times, to ensure consistency of the results. The identity of all PCR

products was verified by DNA sequencing. Controls were treated as

above without adding template and/or reverse transcriptase and

showed no PCR products.

Five microlitres of each PCR product were analysed on 1.5%

agarose gels, stained with ethidium bromide and photographed

using a computer assisted gel documentation system (BioDocAn-

alyse 1.0; Biometra, Goettingen, Germany). Intensities of PCR

signals were determined with the beta 4.0.2 version of the data

acquisition and analysis software from Scion corporation (Scion

Corporation, Frederick, MD, USA). For further data analysis and

graphical output the Microcal Origin 5.5 software (Microcal

Software Inc., Northampton, MA, USA) was used. The statistical

significance of the difference between two data sets was evaluated

with the one-way ANOVA test. Differences were considered signifi-

cant at p < 0.05. All data are expressed as ± SEM.

In order to reliably detect changes in gene expression levels using

semiquantitative RT–PCR techniques, extensive control experiments

were performed. First, to ensure a linear correlation of the original

mRNA concentration with the amount of generated PCR product,

we determined the range of linear amplification for the highly

expressed house keeping gene EF1a (elongation factor 1a) between

15 and 20 PCR cycles under the used conditions (Fig. 1a).

Assuming that gene expression levels for mGluRs are similar or

lower compared to EF1a, subsequent PCR products were generated

using 20 cycles of amplification. Only in those cases, where 20

cycles were not sufficient to generate visible PCR products, 25

cycles were performed.

Table 1 DNA sequence of oligonucleotides

used in this study, species specificity, and

sizes of resulting PCR products (P1 –

sense primer; P2 – antisense primer)

Gene Oligonucleotide [5¢ fi 3¢] Species Size [bp]

EF1a P1-GTCTGCCCAGAAAGCTCAG m + r 291

P2-AATGGTCTCAAAATTCTGTGAC m + r

mGluR1a P1-CTTCAACTCACTTCAGCCCTC m + r 393

P2-GCTCCTCTCGGAAGGTGC m + r

mGluR2 P1-CCACAGAAGAACGTGGTGAG m 159

P1-TACACCACCTGCATCATCTG r 321

P2-TCAAAGCGACGATGTTGTTGAG m + r

mGluR3 P1-CAACCCCAGAAGAATGTGGTC m 156

P1-TACACCACCTGCATCATCTG r 315

P2-TCACAGAGATGAGGTGGTGG m + r

mGluR4a P1-CCGGAGCAGAACGTGCCCA m 145

P2-CTGGGGCCTCCAGGTTCTC m

P1-TGCTCAAGTGCGACATCTCG r 512

P2-CTAGCTGGCATGGTTGGTGTAG r

mGluR4b* P1-CTAATCTTGAGTGTGTTTCGAAG m 179

P2-ACCATCACCAAACACTCAGGC m

P1-TGCTCAAGTGCGACATCTCG r 725

P2-TCAGAGACCATCACCAAACAC r

mGuR5a/b P1-GACTCGGTGGACTCGGGG m + r 129

P2-TCACAACGATGAAGAACTCTG m + r

mGuR6 P1-CATCCAGAGCAGAACGTGC m 99

P1-CCACTTCGCAACTAGTCATC r 554

P2-CTACTTGGCGTCCTCTGAG m + r

mGuR7a* P1-CACCCTGAACTCAATGTCCAG m 198

P1-CTCAAATGTGACATCACAGAC r 510

P2-TTAGATAACCAGGTTATTATAACTG m + r

mGuR8a P1-CATCCAGAGCAGAACGTTCAAAAAC m + r 198

P2-TCAGATTGAATGATTACTGTAGC m + r

mGluR8b* P1-CATCCAGAGCAGAACGTTCAAAAAC m + r 198

P2-TTAGGAAGTGCTCCCGCTC m + r

GABAcR q1 P1-TCTGGGTCAGCTTTGTGTTC m + r 369

P2-TGAATAAAATGTAAGCGGCTGG m + r

GABAcR q2 P1-TCTGGGTCAGCTTTGTGTTC m + r 386

P2-TGAAAACTATGTAGAAGGCAGG m + r

*Mouse ESTs were used to design oligonucleotides specific for mGluR4b (GenBank: AA116337),

mGluR7a (GenBank: BG806878) and mGluR8b (GenBank:BM948607). m, mouse; r, rat; bp, base

pairs.

192 F. M. Dyka et al.


In a second control, the mRNA concentrations of the control gene

EF1a were compared between cDNA templates obtained from the

retinae of DBA/2J mice and C57BL/6 control animals of 2 and

6 months using 20 amplification cycles (Fig. 1b). The calculated

relative intensities were then used to normalize all subsequent PCR

amplifications.

In a third control experiment, different amounts of template

cDNA were correlated with the concentration of the resulting EF1aPCR products, using 20 amplification cycles. Between 0.3 ng and

10 ng, the amount of cDNA correlated in a linear way with the

concentration of the generated PCR products, resulting in a

correlation coefficient of 0.998 (Fig. 1c). Thus, detected differences

in the concentrations of PCR products corresponded in a linear way

with differences in the original mRNA concentrations. In all

subsequent PCR amplifications, 10 ng of cDNA were used.

Finally, we correlated known concentrations of the EF1a PCR

product and our ability to detect these DNA signals using agarose

gel electrophoresis and quantification of the data with the acquisition

and analysis software from Scion corporation (Scion Corporation,

Frederick, MD, USA). Between 0 and 100 ng we observed a linear

relation between DNA concentration and quantification of the

photographed signal, while above 100 ng, our detection system

became saturated (Fig. 1d).

For real time RT–PCR experiments, a LightCycler rapid thermal

cycler system (Roche Diagnostics) and the LightCycler-FastStart

DNA SYBR Green I mix (Roche Diagnostics) were used according

to the manufacturer’s instruction. Specificity of PCR products was

confirmed with melting curve analysis.

Serum deprivation of retinal ganglion cells and RT–PCR

Immortalized rat retinal ganglion cells (RGC-5) were subjected to

serum deprivation as described (Krishnamoorthy et al. 2001).

Briefly, cells were grown in 10-cm dishes in Dulbecco’s modified

Eagle medium (DMEM) supplemented with 100 U/mL penicillin,

100 lg/mL streptomycin and 4 mM L-glutamine in the presence or

absence of 10% fetal calf serum (FCS) for 12–120 h under humified

air at 37�C with 5% CO2 (all chemicals were from Invitrogen).

For RT–PCR cells taken at different time points were washed

three times with PBS and RNAwas isolated using the RNeasy Mini

Kit (Qiagen, Hilden, Germany). Five micrograms of RNA were

reverse-transcribed in a total volume of 20 lL containing 20 U

Superscript II reverse transcriptase, 1 lL 10 mM dNTPs, 4 lL 5·first strand buffer and 250 ng random hexamers (Invitrogen). The

samples were incubated for 15 min at room temperature followed by

2 h at 42�C. Finally, the cDNAwas diluted by the addition of 30 lLsterile water. One microlitre of these samples was amplified as

described above with the following parameters: 3 min at 94�C,

followed by 25–40 cycles with 94�C for 45 s, 62�C for 45 s and

72�C for 60 s, and a final incubation at 72�C for 10 min. Five

microlitres of each PCR product were analysed on 1.5% agarose

gels as described.

Detection of apoptosis in retinal ganglion cells

To detect apoptosis in retinal ganglion cells, RGC-5 cells were grown

on glass coverslips coated with poly-L-lysine (Sigma) and treated as

described with slight modifications (Herkert et al. 2001). Briefly,

cells were washed three times with PBS and fixed on ice in methanol/

acetic acid (95 : 5 v/v) for 30 min. Subsequently, cells were blocked

with 10% sheep serum for 1 h at room temperature. The DNAwas 3¢endlabelled by using terminal desoxynucleotidyl transferase (Prome-

ga, Mannheim, Germany) and TUNEL Label Mix (Roche Diagnos-

tics) for 1 h at 37�C. In parallel, cells were stained for neurofilaments

(NFM – 1 : 500; Chemicon, Hofheim, Germany). After washing

three times with PBS, nuclei were visualized with Hoechst dye

33258 (5 lg/mL; Molecular Probes, Eugene, OR) and the binding

sites of the primary antibody were revealed by a Texas red-

conjugated secondary antibody (1 : 300; Dianova). Cells were rinsed

in PBS, embedded in Mowiol (Sigma) and examined by fluorescence

microscopy (Zeiss Axioscope; Zeiss, Oberkochen, Gemany). Result-

ing images were adjusted in brightness and contrast using Adobe

Photoshop 5.5 (Adobe Systems Inc., San Jose, CA, USA).

Results

DBA/2J mice as a model system for secondary

angle-closure glaucoma

Mice of the DBA/2J strain develop an increased IOP by the

age of 6 months caused by a defect in pigment dispersion,

(a)

(c)

(b)

(d)

Fig. 1 Evaluation of PCR conditions and data analysis. (a) The region

of linear amplification for the house keeping gene EF1a was deter-

mined between 15 and 20 PCR cycles, using 10 ng of retinal cDNA of

6-month-old C57BL/6 control mice as template for each amplification.

(b) Verification of comparable cDNA concentrations between cDNA

samples generated from retinae of DBA/2J mice and C57BL/6 control

animals of 2 and 6 months, using 10 ng of each cDNA type as a

template for the control gene EF1a and 20 amplification cycles. (c)

Linear correlation between different amounts of template cDNA and

the concentration of the resulting PCR products for EF1a, using 20

cycles of amplification. The correlation coefficient for a linear fit of the

data points was calculated as R ¼ 0.998. (d) Correlation between

increasing concentrations of the EF1a PCR product and detected DNA

signals using agarose gel electrophoresis and quantification of the

signals with the acquisition and analysis software from Scion (Scion

Corporation, Frederick, MD, USA). In the entire figure, representative

PCR products are shown in the upper panels, while the lower panels

summarize the data of three experiments. Error bars are ± SEM.



which subsequently results in ganglion cell death, optic nerve

atrophy and optic nerve cupping, and thus are described as a

model for secondary angle-closure glaucoma (John et al.

1998; Chang et al. 1999; Bayer et al. 2001; Anderson et al.

2002; Schuettauf et al. 2002). In order to compare the retinal

gene expression of metabotropic glutamate receptors

(mGluRs) between eyes with normal and elevated IOP, we

used retinae from DBA/2J mice at two different ages. At

2 months we measured an IOP of 6.6 ± 0.9 mmHg, while at

6 months the eye pressure increased to 14.7 ± 3.9 mmHg

(Table 2), which was in agreement with published data

(Savinova et al. 2001). This increase in IOP is followed by

the death of retinal ganglion cells, optic nerve atrophy and

optic nerve cupping leading to blindness (John et al. 1998;

Chang et al. 1999; Schuettauf et al. 2002, 2004).

Metabotropic glutamate receptors are expressed in the

mouse retina and in immortalized ganglion cells of the

rat retina

In a first step, we analysed the expression of mGluR subtypes

and most of their known splice variants in the retina of adult

healthy mice and in an immortalized rat retinal ganglion cell

line, using sequence-specific oligonucleotides (Table 1). The

housekeeping gene elongation factor 1a (EF1a) was used to

ensure equal gene expression between tissues throughout this

study. In addition, we compared the behaviour of mGluRs

with inhibitory GABAC receptors (GABACR) that were

choosen because they are not directly involved in glutamat-

ergic signalling and are predominantly expressed in the retina

(Enz 2001). Using retinal cDNA obtained from adult C57BL/

6 mice that develop no eye disease, we were able to generate

RT–PCR products for all described mRNA types (Fig. 2,

upper panel). In immortalized rat retinal ganglion cells grown

for 48 h in culture, only transcripts for EF1a, mGluR2, both

mGluR5 splice variants and mGluR8b could be detected

(Fig. 2, stars in lower panel). To ensure that the remaining

primer pairs recognizing rat sequences were able to amplify

specific PCR products, we used cDNA obtained from rat

brain and retina (Fig. 2, lower panel).

Expression of metabotropic glutamate receptors is

changed in the retinae of eyes with elevated IOP

To analyse the consequences of elevated IOP for the

expression of mGluR types in the retina, we applied the

above described PCR conditions (see Materials and methods

and Fig. 1) to cDNA obtained from retinae of DBA/2J mice

and C57BL/6 control animals at 2 and 6 months of age,

using sequence-specific primers (Table 1). For most mGluR

subtypes, expression was upregulated between 2 and

Table 2 Intraocular pressure measured in both eyes of DBA/2J mice

at 2 and 6 months

DBA/2J Intraocular pressure (mmHg) Mean (±SD)

2 months

(four animals)

5.2 6.6 ± 0.9

7.6

5.9

7.4

5.9

6.7

7.4

nd

6 months

(five animals)

12.3 14.7 ± 3.9

12.3

15.3

12.8

14.8

13.3

13.3

11.1

24.4

17.8

The data sets between 2- and 6-month-old eyes were significantly

different (p ¼ 0.00007). nd, not determined; SD, standard deviation.

Fig. 2 Metabotropic glutamate receptors expressed in mouse retina

and in ganglion cells of the rat retina. To test the expression of

metabotropic glutamate receptor (mGluR) types in mouse retinae and

rat retinal ganglion cells, RT–PCR products were generated using

cDNA obtained from the retinae of 6-month-old C57BL/6 control mice

(upper panel) or from rat ganglion cells grown for 48 h in culture (stars

in lower panel), using specific primer pairs (see Table 1). If no PCR

product was amplified from the rat ganglion cells, primers were tested

with cDNA obtained from rat retina or brain (lower panel). The house

keeping gene elongation factor 1a (EF1a) and GABAC receptor (GA-

BACR) q-subunits were used as controls in this study. The DNA was

separated on a 1.5% agarose gel and visualized after staining with

ethidium bromide. No PCR products were detected when the reverse

transcriptase was omitted from the cDNA synthesis reaction, verifying

that the cDNA was not contaminated with genomic DNA (not shown).

A 100 bp ladder is indicated on the right. Black and white levels of the

original photograph were inversed for better visualization of the DNA.



6 months within the same species, while mGluR4a,

mGluR4b and mGluR8b were downregulated in the

C57BL/6 control mice (Fig. 3a). All observed differences

were statistically significant (p < 0.05), except for the

changes in DBA/2J mice for mGluR8a (p ¼ 0.42) and in

C57BL/6 animals for mGluR8b (p ¼ 0.32). In order to

balance for slight differences in the concentrations of the four

used cDNA templates, all measured intensities were normal-

ized to the corresponding values of the control PCR product

EF1a (see Fig. 1b). We tested the gene expression of

mGluR7a but not of mGluR7b, as the cDNA sequence of

mGluR7b is only cloned from rat, and a corresponding

mouse EST could not be identified in the database.

To investigate if changes in gene expression levels also

occur in other than glutamate-gated receptor systems, we

analysed two subunits of the GABAC receptor, termed q1and q2. GABAC receptors form GABA gated Cl– channels

and were choosen because they are predominantly expressed

in the retina, where they were localized at synapses of bipolar

cell axon terminals in the inner plexiform layer (Enz 2001).

In contrast to members of the mGluR family, the mRNA

concentration of q1 and q2 were not significantly altered

under elevated IOP (Fig. 3b).

The observed changes in mGluR gene expression could

either be caused by a pathologic change due to the elevated

intraocular eye pressure that develops in DBA/2J mice but

not in the C57BL/6 control animals, or by the normal

development of the animals between 2 and 6 months. To

distinguish between both possibilities, we calculated and

compared the ratios of the observed mRNA concentrations

between 2- and 6-month-old animals for both mouse strains

(Fig. 3c). As mentioned above, changes in gene expression

for mGluR8a (DBA/2J) and mGluR8b (C57BL/6) were not

statistically significant (Fig. 3a). Because the corresponding

ratios were calculated to 1.2 ± 0.17 for mGluR8a and

) 1.1 ± 0.09 for mGluR8b, we classified changes in gene

expression with a ratio smaller than ± 1.5-fold as background

noise (Fig. 3c, dashed horizontal lines).

Comparing the mRNA concentration of 2- and 6-month-

old animals, the increase in retinal gene expression for

mGluR1a, mGluR2, mGluR4a, mGluR4b, mGluR6,

(a)

(b)

(c)

Fig. 3 Gene regulation of metabotropic glutamate receptor types

under elevated IOP. RT–PCR experiments for mGluR types (a) and

GABAC receptor q-subunits (b) were performed using sequence spe-

cific primers (see Table 1) as indicated. cDNA obtained from retinae of

DBA/2J mice of 2 and 6 months of age (representing normal and ele-

vated IOP, respectively), and from C57BL/6 control animals of the

same ages served as templates for the amplifications. For each gene,

representative PCR products are shown in the upper panel, while bar

diagramms summarize measured intensities of PCR products from

three to five experiments. In order to balance for slight differences in

template concentrations between retinal cDNA samples, all measured

intensities were normalized with the corresponding values of the con-

trol PCR product EF1a. The difference in gene expression between

2- and 6-month-old retinae was statistically different (p < 0.05),

except for mGluR8a and GABAC receptor q2 (DBA/2J only), and for

mGluR8b and GABAC receptor q1 (both mouse strains). (c) Bar dia-

gram summarizing calculated ratios in gene expression between reti-

nae of 2- and 6-month-old DBA/2J and C57BL/6 mice. A twofold

increase in mRNA concentration indicates a 100% upregulation of the

corresponding gene. Horizontal dashed lines indicate a 1.5-fold

change in gene expression that was considered not to be significant.



mGluR7a and mGluR8b was higher in DBA/2J animals than

in C57BL/6 control mice (Fig. 3c). In contrast, the relative

increase in transcript levels for mGluR5a/b and mGluR8a

was lower in DBA/2J mice as compared with control

animals, and no differences were noted for the increase of

mGluR3 between both mouse strains. Interestingly, mRNA

levels for mGluR4a, mGluR4b and mGluR8b increased

between 2- and 6-month-old DBA/2J mice, but were

decreased in the control mice. While the calculated ratios

for mGluR8b (DBA/2J ¼ 1.3 ± 0.06; C57BL/6 ¼) 1.1 ± 0.09) are below the threshold of ± 1.5-fold classified

as background noise, ratios for mGluR4b (DBA/2J ¼± 1.4 ± 0.11; C57BL/6 ¼ ) 1.4 ± 0.06) are close to the

threshold. Because the total change in mGluR4b gene

expression is larger than 1.5-fold, we considered this effect

as relevant.

To verify the described changes in mGluR gene expression

obtained by conventional RT–PCR, we used a real-time PCR

system. As before, also for the real-time PCR a series of

control reactions were performed. Between 40 and 1000 pg

of cDNA obtained from the retinae of 2- and 6-month-old

DBA/2J mice, we observed a linear correlation between the

amount of template cDNA and generated EF1a PCR

products, with correlation coefficients of R ¼ ) 0.991 and

R ¼ ) 0.998 (inset of Fig. 4). Thus, for all future amplif-

ications, 200 pg of cDNAwere used as template. In the real-

time PCR method, the amount of generated PCR product is

represented by the CT (cycle of threshold) value that defines

the cycle number at which the amount of the PCR product

exceeded a predetermined threshold. Thus a high mRNA

concentration corresponded to a low CT value, and vice

versa.

Normalized to the values of the EF1a mRNA concentra-

tions, most mGluR subtypes were upregulated between 2 and

6 months in DBA/2J mice (Fig. 4), which was consistent with

our data from the conventional RT–PCR experiments (see

Fig. 3A). Except for mGluR1a, we observed a good qualit-

ative correlation between the data obtained from conventional

and real-time PCR assays, although absolute numbers

differed slightly between the two techniques (Table 3). The

results for the two mGluR8 isoforms are not shown, because

cycle numbers in the real-time PCR were too high for a

reliable calculation (59–62 cycles compared to 27–34 cycles

for all other PCR products). Similarly, the expression of

GABAC receptor q-subunits was not determined by real-time

PCR, as no significant changes were detected by conventional

PCR methods.

In a next step, we compared the distribution of group I

metabotropic glutamate receptors (mGluR1a and mGluR5a/b)

in retinae from eyes with normal and elevated IOP. Staining

vertical sections of 2- and 6-month-old DBA/2J mice with

antibodies specific for mGluR1a or with an immuneserum

recognizing both mGluR5 isoforms revealed prominent

Table 3 Comparison of calculated ratios in gene expression between

2- and 6-month old mice detected by conventional and real-time PCR

methods

Gene

DBA/2J BL/6

Conventional PCR Real-time PCR Conventional PCR

mGluR1a 3.6 ± 0.42 1.2 1.2 ± 0.17

mGluR2 10.3 ± 0.71 7.8 1.5 ± 0.15

mGluR3 2.9 ± 0.29 4.0 2.9 ± 0.24

mGluR4a 3.4 ± 0.29 3.1 ) 1.4 ± 0.12

mGluR4b 1.4 ± 0.11 1.1 ) 1.4 ± 0.06

mGluR5a/b 2.2 ± 0.10 1.5 2.8 ± 0.33

mGluR6 2.9 ± 0.30 2.2 1.4 ± 0.20

mGluR7a 2.6 ± 0.19 1.3 1.9 ± 0.13

mGluR8a 1.2 ± 0.17 nd 2.0 ± 0.06

mGluR8b 1.3 ± 0.06 nd ) 1.1 ± 0.09

GABACRq1 ) 1.0 ± 0.02 nd ) 1.0 ± 0.05

GABACRq2 ) 1.1 ± 0.04 nd ) 1.2 ± 0.03

Values represent x-fold change in mRNA concentration between ret-

inae of 2- and 6-month-old DBA/2J or C57BL/6 control mice. nd, not

determined (see Results for details).

Fig. 4 Real-time PCR for specifically regulated metabotropic glutam-

ate receptors under elevated IOP. The inset shows a linear correlation

between different amounts of cDNA template from DBA/2J mice reti-

nae of 2 and 6 months of age amplified for EF1a, with correlation

coefficients of R ¼ ) 0.991 and R ¼ ) 0.998. The concentration of

PCR products is represented by the cycle of threshold value (CT

value) that defines the PCR cycle number at which the amount of PCR

product exceeded a predetermined threshold. Thus, a high CT value

correspond to a low mRNA concentration, and vice versa. The bar

diagram shows changes in gene expression for mGluR types between

the retinae of DBA/2J mice of 2 and 6 months of age (corresponding to

normal and elevated IOP). Values were normalized to those of the

corresponding EF1a control reactions and calculated according to the

software of the LightCycler system.



expression of these receptors in the inner plexiform layer (IPL)

and outer plexiform layer (OPL) of the retinae (Fig. 5). In

addition, occasional labelling of cell bodies was seen in the

inner nuclear layer (INL). At 6 months, somata in the

ganglion cell layer (GCL) were surrounded with mGluR1a

(arrows in Fig. 5b), which was not detected in the 2-month-

old animals. Moreover, increased immunofluorescence for

mGluR5a/b was observed in both plexiform layers under

elevated IOP, and cell bodies in the GCL appeared brighter

(arrows in Fig. 5c). The increase in fluorescence in the

plexiform layers and in the GCL suggested a higher protein

concentration at elevated IOP for mGluR1a and mGluR5a/b

at these locations. As already noted by others (Bayer et al.

2001), we always observed a thinning of the retinal layers

in the 6-month-old retinae compared to the younger animals

at the same eccentricity.

Retinal ganglion cells upregulate metabotropic glutamate

receptors under apoptotic conditions

Increased IOP in 6-month-old DBA/2J mice results in

apoptotic cell death of retinal ganglion cells (John et al.

1998; Chang et al. 1999; Bayer et al. 2001; Anderson et al.

2002; Schuettauf et al. 2002, 2004). To investigate, if the

observed gene regulation of mGluRs in the retinae of these

mice was caused by apoptotic mechanisms, we used an

immortalized rat retinal ganglion cell line (RGC-5; Krishna-

moorthy et al. 2001). In these cells, we detected the

expression of mGluR2, both mGluR5 isoforms, and

mGluR8b by RT–PCR (stars in Fig. 2b). PCR products for

other mGluR isoforms and for GABAC receptor q-subunitscould be amplified from whole rat retinae or brain tissue, but

not from the ganglion cell line (Fig. 2b).

A prominent hypothesis explaining a possible mechanism

of ganglion cell death in glaucoma is mechanical squeezing

by an increased IOP of the lamina cribrosa of the optic nerve

head, the region where the axons of ganglion cells leave the

eye (Morgan 2000). Due to this mechanical force, the

retrograde transport of nerve growth factors from the axon

terminals to the ganglion cell somata may be reduced,

causing ganglion cells to initiate apoptotic processes. To

mimic this effect in cell culture, we induced apoptosis in

RGC-5 cells by withdrawl of fetal calf serum which contains

nerve growth factors, and monitored the process of apoptosis

by a TUNEL assay at different time points. Nuclei were

(a) (b) (c)

Fig. 5 Immunohistochemical comparison of metabotropic glutamate

receptors in DBA/2J mice. (a) The retinal layers are shown with

Nomarski optics: ONL, outer nuclear layer; OPL, outer plexiform layer;

INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell

layer. (b) Fluorescence micrographs of vertical cryostat sections

through the retinae of 2- and 6-month-old DBA/2J mice, incubated with

antibodies specific for mGluR1a. The upper two panels show expres-

sion of mGluR1a in the OPL and the IPL, and occasional staining of

somata of the INL. In 6-month-old retinae cell bodies in the GCL were

labeled (arrows). This is better seen in the two lower panels showing

regions of the IPL and the GCL in higher magnification. (c) Fluores-

cence micrographs stained for mGluR5a/b as described in (b). At

6 months the fluorescence was increased in both plexiform layers, and

somata in the GCL were labelled more intensly (arrows). Additional

staining was detected in the INL. Scale bars are 20 lM.



visualized using Hoechst 33258 dye and the shape of the

ganglion cells was evident from staining for neurofilaments.

The lack of fetal calf serum induced apoptosis already after

12 h, which reached a plateau between 24 h and 48 h, as

estimated by optical inspection of the stained cells (Fig. 6, –

FCS). Cells grown under control conditions did not show any

apoptotic events till 48 h in culture (Fig. 6, + FCS).

To monitor the time course of gene expression for mGluRs

detectable in these ganglion cells (see stars in the lower panel

of Fig. 2), RNA was isolated at different time points and

subjected to RT–PCR. During apoptosis, the concentration of

the transcript for EF1a remained nearly constant (Fig. 7a). In

contrast, mGluR2 was strongly upregulated after 48 h in

culture under apoptotic conditions (Fig. 7b), while a lower

but significant upregulation of mGluR5a/b occured already

after 24 h (Fig. 7c). On the other hand, mGluR8b did not

show any significant changes (Fig. 7d). These observations

are consistent with our data from mice retinae that showed a

strong upregulation of mGluR2 and a weaker increase of

mGluR5a/b under elevated IOP, while mGluR8b was not

significantly changed (Fig. 3).

Discussion

In this study we analysed changes in the gene expression of

metabotropic glutamate receptors in retinae of a mouse

model for secondary angle-closure glaucoma (DBA/2J; John

et al. 1998; Chang et al. 1999; Bayer et al. 2001; Anderson

et al. 2002; Schuettauf et al. 2002), and compared these data

with ganglion cells of the rat retina undergoing apoptosis. To

gain insight into the development of the pathomechanisms

leading ultimatively to ganglion cell death, we used retinae

of DBA/2J mice at 2 and 6 months of age. These time points

were choosen, because between 2 and 6 months of age we

Fig. 6 Detection of apoptosis in a ganglion cell line from rat retina. RGC-5 cells were grown in the absence (– FCS) and presence (+ FCS) of fetal

calf serum for the indicated time periods. Apoptosis was analysed by DNA fragmentation in nuclei stained by Hoechst dye 33258 (blue) using a

TUNEL assay (green). The shape of the cells was visualized by an antiserum against neurofilament M (NFM, red).



detected an increase in IOP of about twofold, from 6.6 ± 0.9

to 14.7 ± 3.9 mmHg (Table 2). To ensure that differences in

gene expression between 2- and 6-month-old retinae were

indeed due to the elevated IOP, and not caused by

developmental processes of the animals, we used retinae of

2- and 6-month-old C57BL/6 mice as controls that do not

develop eye diseases.

To compare the gene expression in the above-described

retinae, conventional semiquantitative RT–PCR techniques

were combined with the real-time PCR method and immu-

nohistochemistry. We found mRNA concentrations of

mGluR1a, mGluR2, mGluR4a, mGluR4b, mGluR6 and

mGluR7a significantly increased under elevated IOP in the

retinae of 6-month-old DBA/2J mice, when compared to

2-month-old animals of the same strain and to C57BL/6

control animals. At 6 months, no significant death of

ganglion cells was described (John et al. 1998; Schuettauf

et al. 2002), thus detected differences in gene expression are

most likely due to the adaptation of molecular mechanisms to

the pathological process in the retina, rather than caused by a

loss of ganglion cells, which occurs at older ages. In contrast

to mGluR types, the expression levels of inhibitory GABAC

receptor subunits did not change under these conditions.

Although we did not test the gene expression levels of other

inhibitory GABA and glycine receptor subunits, this finding

indicated that increased IOP induces major changes in the

expression level of the glutamatergic system, while inhibi-

tory acting receptors seem not to be affected to the same

extend. This hypothesis points to an involvement of the

neurotransmitter glutamate in the observed changes in gene

regulation, and indeed a downregulation of a glutamate

transporter was observed in the retinae of glaucoma patients

(Naskar et al. 2000).

When comparing 2-month-old retinae of DBA/2J and

C57BL/6 animals, we observed different expression levels of

some mGluR types (e.g. mGluR4a). While it seems likely

that this finding is an inherited characteristic of the different

mouse strains, it is not clear whether the observed differences

reflect a predisposition for retinal degeneration in the DBA/2J

mice.

Our data are in agreement with a previous study that

analysed the expression pattern of mGluR types in ganglion

cells of the adult rat retina after axotomy of the optic nerve

(Tehrani et al. 2000). Upon cutting the axons of the ganglion

cells, the authors observed an upregulation of mGluR1a,

mGluR6 and mGluR7a by single-cell RT–PCR, consistent

with our findings. Cutting axons of ganglion cells might be

regarded as an extreme consequence of mechanical squeez-

ing at the optic nerve head, caused by increased IOP.

Therefore, in both situations ganglion cells might initiate

apoptotic pathways, which could explain the upregulation of

mGluR1a, mGluR6 and mGluR7a under both conditions.

However, some differences exist, as evident from the

behaviour of mGluR2 and mGluR4a. While their gene

expression was not changed significantly upon optic nerve

axotomy (Tehrani et al. 2000), we observed a strong increase

in transcript levels under elevated IOP or in cell culture

(a)

(b)

(c)

(d)

Fig. 7 Metabotropic glutamate receptors are upregulated in ganglion

cells under apoptotic conditions. RT–PCR experiments for EF1a (a),

mGluR2 (b), mGluR5a/b (c) and mGluR8b (d) were performed using

sequence specific primers (see Table 1), as indicated. cDNA obtained

from retinal ganglion cells grown for the indicated time periods in the

absence (– FCS) and presence (+ FCS) of fetal calf serum served as

templates for the amplifications. Each bar summerizes the intensities

of PCR products from three to five experiments. In order to balance for

slight differences in template concentrations between retinal cDNA

samples, all measured intensities were normalized with the corres-

ponding value of the control PCR product EF1a. Stars indicate a

statistical difference in gene expression of p < 0.05 compared to

control cells at 48 h (+ FCS).



inducing apoptosis. If these differences are due to the

different stimuli applied in both studies, or simply caused by

the use of different model systems (rat, mouse and cell lines)

remains to be clarified.

Although ganglion cell loss under elevated IOP is the primary

observation in DBA/2J mice, this does not exclude adaptation

processes elsewhere in the retina, or as a direct reaction of the

elevated pressure, or as a consequence of ganglion cell death.

Therefore, the expression of proteins in cell types other than

ganglion cells could also be changed under pathologic condi-

tions. To find out which cell types in the retina participated in the

observed upregulation of mGluRs, we focussed on the group I

metabotropic glutamate receptors mGluR1a and mGluR5a/b.

Staining of vertical cryostate sections of mice retina from 2- and

6-month-old DBA/2J mice revealed prominent expression of

mGluR1a and mGluR5a/b in the two plexiform layers of the

retina, while cell bodies in the INLwere sparsely labelled, which

was consistent with the literature (Koulen et al. 1997). However,

in the retina of 6-month-old DBA/2J mice, both immunesera

strongly stained somata in the GCL, which was not observed

under healthy conditions, and therefore might be caused by the

elevated IOP in these animals. Similar to our finding, transcripts

formGluR6were only detectable in ganglion cells after axotomy

of adult rats (Tehrani et al. 2000), indicating that injury to

ganglion cells can cause an abnormal upregulation of mGluR

types. Thus, the observed increase in transcript level of those

mGluR types not analysed by immunocytochemistry (e.g.

mGluR6) might result from a pathological gene regulation in

ganglion cells. On the other hand, upregulation of the receptors

might also occur in those neurones that express the proteins

under normal conditions. In the case of mGluR6, this would

indicate an involvement of bipolar cells in the glaucomateous

process of 6-month-old DBA/2J animals, because only rod

bipolar cells express mGluR6 in the adult retina (Masu et al.

1995; Vardi et al. 2000).

MGluR5a/b appeared significantly increased in both

plexiform layers of 6-month-old DBA/2J mice. Ganglion,

amacrine and bipolar cells form synapses in the IPL, while

the OPL is composed of contacts between photoreceptors,

bipolar and horizontal cells. Thus, stronger labelling in the

IPL indicated an upregulation of the mGluR5 isoforms in

ganglion cells, but possibly also in amacrine and bipolar

cells. Increased fluorescence in the OPL suggested an

upregulation of mGluR5a/b in bipolar cells, as horizontal

cells and photoreceptors do not express mGluR5 (Koulen

et al. 1997). However, we cannot exclude abormal expres-

sion of mGluR5 isoforms in horizontal cells and photore-

ceptors under pathological conditions of 6-month-old DBA/2J

mice. Somata of photoreceptors, located in the ONL, never

showed specific staining for mGluR1a and mGluR5a/b, in

agreement with the absence of these proteins in photorecep-

tors (Koulen et al. 1997). Furthermore, photoreceptors do

not seem to be affected by the retinal degeneration in older

DBA/2J mice (Schuettauf et al. 2004).

To test if the observed changes in the gene regulation of

glaucomateous mouse retinae were due to apoptotic mech-

anisms, we used a rat retinal ganglion cell line expressing a

subset of mGluR isoforms (Krishnamoorthy et al. 2001).

Because the mGluR expression pattern as well as the

morphology of the cell culture differed from normal retinal

ganglion cells (see next paragraph), this cell culture is not a

substitute for an in vivo situation. On the other hand,

expression of mGluR1a and mGluR5a/b, but not of mGluR6,

in ganglion cells of the adult rat is in agreement with other

studies (Tehrani et al. 2000). Therefore, we induced apop-

tosis in this cell line by serum deprivation, which resulted in

a strong upregulation of mGluR2, and a smaller effect for

mGluR5a/b, while no significant change was observed for

mGluR8b. Withdrawl of fetal calf serum in cell culture

mimics, but is not identical to the hypothesized reduction of

the retrograde transport of nerve growth factors from the

axon terminals to the ganglion cell somata, which is

discussed as a possible mechanism of ganglion cell death

in glaucoma. Thus, we cannot exclude that different

molecular mechanisms are triggered in cell culture and in

the DBA/2J mice. However, observed changes in the

expression of mGluR2 and mGluR5a/b in cell culture are

in agreement with the data obtained from mice retina,

suggesting that similar molecular pathways may be involved

under both conditions. Future experiments have to show if

these changes are the cause or a result of apoptotic

mechanisms occuring in the ganglion cell culture.

Most of the investigated mGluRs were previously detected

in neurones of the mammalian retina. In the above-discussed

single-cell RT–PCR study (Tehrani et al. 2000), transcripts

for mGluR1–8 could be amplified from individual ganglion

cells of juvenile and/or adult rat retinae. Using in situ

hybridization techniques, mGluR1a, mGluR2, mGluR4 and

mGluR7a were present in the ganglion cell layer of adult rat

retinae (Hartveit et al. 1995). With receptor specific im-

munesera, mGluR8 was observed on somata and dendrites of

ganglion cells (Koulen and Brandstatter 2002), and mGluR2,

mGluR3 and mGluR5a/b were detected along their axons

(Jeffery et al. 1996), a cell compartment heavily affected in

glaucoma. It was hypothesized that increased IOP disrupts

the function of retinal ganglion cell axons by increasing

mechanical forces on the lamina cribrosa of the optic nerve

head (Morgan 2000). Alternatively, astrocyte–axon interac-

tions between astrocytes of the optic nerve and ganglion cell

axons may be important. However, localization of receptor

proteins in axons could simply represent axonal transport to

terminal systems, rather than indicating functional receptors

localized along the axon.

Besides the metabotropic glutamate receptor system,

changes in gene expression under increased IOP were also

observed for ionotropic glutamate receptors and for glutam-

ate transporters. A recent study described the downregulation

of the glutamate transporter EAAT-1 in retinal Muller cells as



a potential mechanism leading to elevated concentrations of

the excitatory neurotransmitter glutamate at glutamatergic

synapses, which ultimately results in the death of ganglion

cells in the retinae of glaucoma patients (Naskar et al. 2000).

Indeed, a reduction of retinal glutamate transporter function

resulted in elevated glutamate levels and subsequent gan-

glion cell death (Vorwerk et al. 2000). Furthermore, elevated

glutamate concentrations reduced the amount of the NMDA

receptor subunit 1, interpreted as a compensatory mechanism

for the increased glutamate levels (Naskar et al. 2000).

However, another group did not find significant changes in

NMDA receptor expression in experimentally induced

glaucoma of monkey eyes (Hof et al. 1998).

In summary, we found most metabotropic glutamate

receptors significantly changed under elevated IOP, while

inhibitory GABAC receptors seemed not to be affected. In

contrast, it has been shown that NMDA receptor subunits were

downregulated in excitotoxic disease states, most likely to

compensate elevated concentrations of glutamate (Piehl et al.

1995; Kreutz et al. 1998). Currently, it is not clear whether the

observed changes in mGluR gene expression trigger apoptotic

mechanisms in retinal ganglion cells, or if they are a

consequence of this process. As 6-month-old DBA/2J mice

do not show a significant loss of ganglion cells (John et al.

1998; Schuettauf et al. 2002), one can assume that differences

in gene expression at this age are most likely involved in the

pathological process of the retina, and not a secondary effect

caused by dying ganglion cells. Upregulation of mGluR types

can influence the intracellular concentration of second mes-

sengers: mGluR1 and mGluR5 activate phospholipase-C and

thus trigger the release of Ca2+ from intracellular stores, while

all other mGluR types are negatively coupled to adenylyl

cyclase (Conn and Pin 1997). If these processes actively

initiate apoptotic pathways in ganglion cells of glaucomateous

retinae, or if they passively adapt to a changed environment,

seems to be a prior direction for future research.

Acknowledgements

We thank Andreas Ohlmann for providing the DBA/2J mice, Neeraj

Agarwal for the kind gift of RGC-5 ganglion cells, Gabi Sass for

help with the PCR light-cycler, Erika Jung-Korner and Nadja

Schroder for excellent technical assistance, Adaling Ogilvie and

Cord-Michael Becker for support, and Pamela Strissel for critically

reading the manuscript. This work was supported by the Deutsche

Forschungsgemeinschaft (SFB539).

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metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure

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