q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390 383

Journal of Neurochemistry, 2001, 77, 383±390

Increases in cortical glutamate concentrations in transgenic

amyotrophic lateral sclerosis mice are attenuated by creatine

supplementation

Ole A. Andreassen,* Bruce G. Jenkins,² Alpaslan Dedeoglu,* Kimberly L. Ferrante,*Mikhail B. Bogdanov,* Rima Kaddurah-Daouk,³ and M. Flint Beal*,§

*Neurochemistry Laboratory, Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston,

Massachusetts, USA

²Department of Radiology, MGH-NMR Center, Massachusetts General Hospital and Harvard Medical School, Boston,

Massachusetts, USA

³Avicena Group, Cambridge, Massachusetts, USA

§Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York Presbyterian Hospital,

New York, USA

Abstract

Several lines of evidence implicate excitotoxic mechanisms in

the pathogenesis of amyotrophic lateral sclerosis (ALS).

Transgenic mice with a superoxide dismutase mutation

(G93A) have been utilized as an animal model of familial

ALS (FALS). We examined the cortical concentrations of

glutamate using in vivo microdialysis and in vivo nuclear

magnetic resonance (NMR) spectroscopy, and the effect of

long-term creatine supplementation. NMDA-stimulated and

L-trans-pyrrolidine-2,4-dicarboxylate (LTPD)-induced increases

in glutamate were signi®cantly higher in G93A mice compared

with littermate wild-type mice at 115 days of age. At this age,

the tissue concentrations of glutamate were also signi®cantly

increased as measured with NMR spectroscopy. Creatine

signi®cantly increased longevity and motor performance of the

G93A mice, and signi®cantly attenuated the increases in

glutamate measured with spectroscopy at 75 days of age, but

had no effect at 115 days of age. These results are consistent

with impaired glutamate transport in G93A transgenic mice.

The bene®cial effect of creatine may be partially mediated by

improved function of the glutamate transporter, which has a

high demand for energy and is susceptible to oxidative stress.

Keywords: creatine, glutamate, glutamate transport, oxida-

tive damage, magnetic resonance spectroscopy.

J. Neurochem. (2001) 77, 383±390.

Amyotrophic lateral sclerosis (ALS) is a severe neuro-

degenerative disorder characterized by motor neuron loss,

rapidly progressive motor weakness and early death (Brown

1995). A role for excitotoxicity in the pathogenesis of ALS

has been suggested. An increase in CSF levels of excitatory

amino acids, as well as a decrease in synaptosomal high-

af®nity Na1-dependent glutamate uptake in spinal cord was

reported (Rothstein et al. 1990; Rothstein et al. 1992). This

reduction was subsequently ascribed to reduced levels of

EAAT2 protein, the major astroglial glutamate transporter

(Rothstein et al. 1995; Lin et al. 1998). There was no alteration

in mRNA expression for glutamate transporters (Bristol and

Rothstein 1996), however, there was abnormal splicing of

EAAT2 mRNA transcripts in 65% of sporadic ALS patients,

but not in controls (Lin et al. 1998). Abnormal splicing in

normal controls however, may also occur (Meyer et al. 1998;

Received August 18, 2000; revised manuscript received October 31,

2000; accepted November 14, 2000.

Address correspondence and reprint requests to M. Flint Beal,

M.D., Chairman, Department of Neurology and Neuroscience,

New York Presbyterian Hospital/Weill Medical College of Cornell

University, 525 East 68th Street, New York, NY 10021, USA.

E-mail: fbeal@mail.med.cornell.edu

Abbreviations used: ALS, amyotrophic lateral sclerosis; FALS,

familial ALS; Glx, combined glutamine and glutamate; LTPD,

l-trans-pyrrolidine-2,4-dicarboxylate; 2-ME, 2-mercaptoethanol; OPA,

o-pthalaldehyde; PCR, polymerase chain reaction; SOD, superoxide

dismutase.

Honig et al. 2000). There were no mutations in the EAAT2

gene to account for aberrant splicing (Aoki et al. 1998; Meyer

et al. 1998). In organotypic spinal cord cultures, inhibitors of

glutamate transport cause degeneration of motor neurons

(Rothstein et al. 1993), and administration of antisense oligo-

nucleotides to EAAT2 leads to neurodegeneration in the spinal

cord and progressive paralysis in vivo (Rothstein et al. 1996).

Other evidence for excitotoxicity was the ®nding that

human motor neurons preferentially express calcium per-

meable AMPA receptors (Williams et al. 1997). Activation

of calcium-permeable AMPA receptors can produce selec-

tive death of motor neurons in spinal cord (Roy et al. 1998;

Terro et al. 1998; Carriedo et al. 2000). Activation of

calcium-permeable AMPA receptors in motor neurons leads

to an accumulation of mitochondrial calcium, mitochondrial

depolarization, a preferential ¯ux of Zn21 and the genera-

tion of reactive oxygen species (Sensi et al. 1999; Carriedo

et al. 2000). The calcium permeability of AMPA receptors

depends on post-transcriptional RNA editing, which changes

a glutamine to an arginine in the GluR2 subunit. The

expression of the GluR2 subunit is reduced in ALS and the

editing ef®ciency was signi®cantly reduced in spinal cord of

ALS cases (Takuma et al. 1999).

The discovery that autosomal dominant familial ALS is

associated with point mutations in the enzyme Cu/Zn super-

oxide dismutase (Rosen et al. 1993), led to the development

of transgenic mouse models of ALS (Gurney et al. 1994;

Wong et al. 1995; Bruijn et al. 1997). These mice show

several similarities to the human disease, including rapidly

progressive motor weakness from 100 days of age, and

premature death at around 135 days of age. There are

increased markers of oxidative stress in the motor neurons

of the G93A mice (Ferrante et al. 1997), and levels of

oxidative markers are increased in vivo as detected using

microdialysis in the striatum in freely moving animals

(Bogdanov et al. 1998). The phenotype of the G93A mice is

exacerbated by a partial de®ciency of manganese superoxide

dismutase, which further supports the involvement of

oxidative stress (Andreassen et al. 2000).

Using the transgenic ALS mouse model, Canton and

coworkers (Canton et al. 1998) showed reduced uptake of

glutamate in synaptosomal preparations from the spinal cord

of endstage ALS mice. Another study showed impaired

cortical synaptosomal uptake at three months of age (Guo

et al. 2000). A recent study of end-stage ALS mice showed

elevated levels of glutamate and aspartate as well as reduced

glutamate extraction in cortical microdialysates (Alexander

et al. 2000). In the present study we examined whether

glutamate levels are increased in transgenic ALS G93A

mice using both in vivo microdialysis and NMR spectro-

scopy. We investigated the effect of creatine supplemen-

tation in these paradigms, as we have previously shown a

signi®cant improvement in survival with creatine adminis-

tration (Klivenyi et al. 1999).

Materials and methods

Mice

Transgenic male mice with the G93A human SOD1 (G1H/1)

mutation (B6SJL-TgN (SOD1-G93A)1 Gur; Jackson Laboratories,

Bar Harbor, ME, USA) were bred with female B6SJL mice

(Jackson Laboratories). The F1 generations were genotyped with

polymerase chain reaction (PCR) on tail DNA and used in the

experiments. All animal experiments were carried out in accord-

ance with the NIH Guide for the Care and Use of Laboratory

Animals and were approved by the local animal care committee.

Treatment/protocol

Creatine (Avicena Group, Cambridge, MA USA) was mixed into

the mouse food (Purina Test Diet, Richmond, IN USA) at 2%

concentration. Treatment started at 4 weeks of age. At 80 days of

age, animals were selected for NMR spectroscopy, and the same

animals were re-used for spectroscopy at 110 days of age. Animals

for in vivo microdialysis were killed after the experiments at 75 and

110 days of age. Twelve 2% creatine-fed and 14 unsupplemented

G93A mice were followed until end stage for survival studies.

Another group of mice were treated with 1%, 2% and 3% creatine

(12±14 mice/group) and followed for survival.

Microdialysis

Non-metallic concentric microdialysis probes (membrane length

2 mm, outer diameter 220 mm; molecular weight cut-off 13.5 kDa;

Spectrum Inc., Houston, TX USA; recovery for glutamate in vitro

10±12%) were implanted into the right prefrontal cortex with the

animal under halothane anesthesia. Coordinates from bregma: AP

2.4, ML 1.2, V 3.2. Animals then recovered for 20±24 h before the

microdialysis experiments. The microdialysis ¯uid path consisted

of either fused silica (Polymicro Technologies, Inc., Phoenix, AZ

USA) or polyetherketone (PEEK, Bioanalytical Systems, West

Lafayette, IN, USA). All metal parts were passivated with 6 m

nitric acid. During the microdialysis animals were freely moving in

a plastic cage. The mice were perfused at 2 mL/min (Microdialysis

pump Model 102 CMA, Microdialysis, Acton, MA USA) with

arti®cial CSF composed of 145 mm NaCl, 2.7 mm KCl, 1.0 mm

MgCl2, 1.2 mm CaCl2, pH 7.4. After an equilibrium period of at

least 1.5 h, dialysates were collected every 20 min, and the ®rst

four were used as baseline samples. Mice were then perfused for

20 min with 0.50 mm of N-methyl-d-aspartate (NMDA, Sigma,

St Louis, MO, USA), or with high K1 concentration (47.7 mm

NaCl, 100 mm KCl, 1.0 mm MgCl2, 1.2 mm CaCl2, pH 7.4), or for

40 min with 1.0 mm l-trans-pyrrolidine-2,4-dicarboxylate (LTPD;

pyrrolidine, Sigma). After the stimulation period, dialysates were

collected every 20 min for a total of six samples. After completion

of the experiments, all mice were killed and the location of the

probes were veri®ed.

Glutamate measurements

Concentrations of glutamate in microdialysates were detected by

HPLC after precolumn derivatization with o-pthalaldehyde (OPA).

The derivatization stock reagent contained 27 mg of OPA, 1 mL of

100% methanol, 10 mL of 2-mercaptoethanol (2-ME) and 9 mL of

0.1 m sodium tetraborate, pH 9.3. The working solution was pre-

pared by diluting the stock solution 1 : 10 with 0.1 m sodium

tetraborate. Derivatization was performed by mixing 10 mL of the

sample or standard with 10 mL of the working OPA/2-ME solution

384 O. A. Andreassen et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390

for 2 min at 1 248C. Ten microliters of the mixture was injected on

the column. Analysis was performed using gradient reverse-phased

HPLC on a four-channel (300, 400, 640 and 660 mV), coulometric

array system (CoulArray, Model 5600, ESA, Chelmsford, MA

USA). Analytes were separated on a C18 ODS 3 mm 80 � 4.6 mm

column (HR-80, ESA, Chelmsford, MA USA) at 1 mL/min. Mobile

phase A was 100 mm Na2HPO4, pH 6.6, 20% (v/v) methanol;

mobile phase B was 100 mm Na2HPO4, pH 6.6, 20% (v/v)

methanol, 15% (v/v) acetonitrile. The gradient pro®le was as

follows: 0% B for 2 min, a linear increase to 100% B over 2 min,

hold for 4 min, return to 0% B over 1 min. The temperature of the

column and detectors was set at 328C.

NMR spectroscopy

Magnetic resonance spectroscopy was run on a 4.7-T GE Omega

(Fremont, CA USA) imager in the FALS G93A mice between

75 and 118 days of age. Spectra were collected using a PRESS

sequence with TR 2 s and TE 136 ms as described (Jenkins et al.

2000). The average voxel size was approximately 40 mL

(, 6.5 � 1.8 � 3 mm) covering sensorimotor cortex bilaterally.

Mice were imaged under halothane/N2O/O2 anesthesia (1.5% halo-

thane), and were maintained at 388C using a circulating water

blanket. The data were analyzed by integration of peak areas ®tting

the peaks to mixed Lorenzian±Gaussian lineshapes. Values were

determined for N-acetylaspartate and creatine using choline as a

standard. Normalization of the choline values to water spectra from

the same voxel (TR/TE 10 s/10 ms) indicated no signi®cant

difference between the choline values in the creatine-supplemented

and the non-supplemented mice.

Behavior and weight

The motor performance was observed in 12±15 animals from each

treatment group twice weekly from 70 days of age after one week

to get acquainted to the rotarod apparatus (Columbus Instrument,

Columbus, OH, USA). During testing, the mice were placed on a

rod that rotated for 12 r.p.m., and the time until they fell of the rod

was used as the measure of the competence on the task. Maximum

score was 60 s, each mouse had three trials and the best result was

recorded. The weights were recorded once a week.

Survival

The initial sign of the disease was a resting tremor, which pro-

gressed to gait abnormalities, paralysis of the hind limbs and at

end stage a nearly complete paralysis. The animals were killed

when they were no longer able to groom or were unable to roll over

within 10 s after being pushed to their side. This time point was

taken as the time of death.

Statistics

Data are expressed as the mean ^ standard error of the mean.

Statistical comparisons of the spectroscopy data were by Student's

t-test, and the behavior and microdialysis results were analyzed by

analysis of variance (anova). The Mantel±Cox test was used to

analyze survival.

Results

The effects of the creatine supplementation on motor per-

formance, weight loss and survival are shown in Figs 1, 2

and 3, respectively. Creatine 2% signi®cantly increased

survival by 14.6% in G93A mice, from 133.6 ^ 2.5 in

the regular fed to 153.2 ^ 2.8 in the treated mice (mean

^ SEM, p , 0.001, Mantel±Cox test). The motor perfor-

mance in G93A mice on unsupplemented diets started to

decline at age 110 days, and 2% creatine treatment delayed

the onset of motor de®cits by approximately 15 days. It

signi®cantly improved the rotarod performance from 114

days of age (114±149 days, p , 0.02). The weight loss

in G93A mice was also delayed by 2% creatine in the

diet. The unsupplemented mice had a signi®cantly lower

weight as compared with creatine fed mice from age 125

days and older ( p , 0.05, Fig. 2). We carried out a further

Fig. 1 Effects of 2% creatine supplementation on rotarod perfor-

mance in G93A transgenic mice. As compared with wild-type mice,

the G93A mice showed impaired rotarod performance that was sig-

ni®cantly attenuated by creatine supplementation between 116 and

140 days of age. *p , 0.05, compared with G93A mice fed normal

diets (n � 12±14/group). X, Control; W, 2% creatine; P, wild type.

Fig. 2 Effects of 2% creatine supplementation on weight loss in

G93A transgenic mice. As compared with wild-type mice, there was

a progressive weight loss by 108 days of age that was signi®cantly

attenuated by creatine. *p , 0.05 compared with G93A mice fed

normal diets (n � 12±14/group). X, Control; W, 2% creatine; P, wild

type.

Creatine effects on glutamate in ALS mice 385

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390

experiment to examine whether 3% creatine would result in

improved survival, as compared with 1% and 2% creatine.

As shown in Fig. 4, the administration of 1% and 3%

creatine were not as ef®cacious as 2% creatine in improving

survival.

An NMR spectrum from a littermate wild-type mouse, a

75-day-old G93A on an unsupplemented diet and a 2%

creatine mouse is shown in Fig. 5. The results of the NMR

spectroscopy are presented in Table 1. At 75 days of age

there was a signi®cant increase in Glx (combined glutamine

and glutamate) concentration in G93A mice, as compared

with wild-type controls ( p , 0.01). Administration of 2%

creatine signi®cantly increased the Cr/Cho level ( p , 0.05)

(Table 2). The temporal evolution of the changes in NMR

Fig. 3 Effects of 2% creatine supplementation on cumulative survi-

val in G93A transgenic mice. Creatine supplementation signi®cantly

improved survival ( p , 0.001; n � 12±14/group). X, Control; W, 2%

creatine.

Fig. 4 Effects of 1%, 2% and 3% creatine supplementation on

mean survival in G93A transgenic mice. All doses of creatine

resulted in a signi®cant improvement of survival, but the effects of

2% creatine were better than with either 1% or 3% creatine

(n � 12±14/group).

Fig. 5 Left-hand panel: two proton spectra

from sensorimotor cortex in a wild-type and

FALS mouse plotted on top of one another.

The spectra are nearly identical except for

a large increase in the Glx (glutamate 1

glutamine) peak in the FALS mouse. The

other abbreviations are: taurine (Tau);

cholines (Cho); creatine 1 phosphocreatine

(Cr); N-acetylaspartate (NAA). Right-hand

panel: comparison of spectra from a crea-

tine-fed and normal-diet FALS mouse. Note

the large decrease in the Glx peak in the

creatine-fed animal.

Table 1 Comparison of wild-type and FALS mice for metabolite levels

Chemical

FALS (normal diet)

(n � 9)

wild type (normal diet)

(n � 6)

NAA/Cr 1.38 �̂ 0.25 1.44 �^ 0.36

Cho/Cr 0.92 �̂ 0.14 0.82 �^ 0.17

Glx/Cr 0.56 �̂ 0.19 0.28 �^ 0.11 ( p , 0.01)

Glx � glutamate 1 glutamine.

Table 2 Comparison of FALS normal diet with FALS 2% creatine diet

Chemical

FALS (normal diet)

(n � 9)

FALS (2% cr diet)

(n � 6)

NAA/Cho 1.53 �^ 0.31 1.63 �̂ 0.17

Cr/Cho 1.11 �̂ 0.15 1.31 �̂ 0.18 ( p , 0.02)

Glx/Cho 0.60 �̂ 0.16 0.49 �̂ 0.23

386 O. A. Andreassen et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390

spectroscopy in ®ve mice on normal vs. 2% creatine diets

are shown in Table 3. There was a signi®cantly higher Glx

level at both 80 and 110 days of age in the G93A mice

( p , 0.02), and creatine signi®cantly reduced the Glx peak

at 80 days of age.

The results of the microdialysis experiments at 110 days

of age are shown in Figs 6, 7 and 8. NMDA signi®cantly

increased the glutamate concentration from baseline levels,

but the increase was nearly twice as high in the G93A mice,

with a signi®cantly higher glutamate concentration as com-

pared with littermate control mice at time points 20 and

40 min after local NMDA application (Fig. 6, p , 0.01).

The high [K1] application signi®cantly elevated the

glutamate levels from baseline, but here the response in

Table 3 Temporal evolution of metabolite changes in normal diet and 2% creatine diet FALS mice

NAA/Cho Cr/Cho Glx/Cho

ALS 2 normal diet

1st time point (80 days; n � 5) 1.56 �^ 0.57 1.08 �^ 0.19 0.62 �̂ 0.24

2nd time point (110 days, same animals) 1.65 �^ 0.27 1.24 �^ 0.11 0.56 �^ 0.15

ALS 2 creatine diet

1st time point (80 days; n � 5) 1.47 �^ 0.2 1.07 �^ 0.22 0.27 �̂ 0.15 ( p , 0.03)*

2nd time point (110 days, same animals) 1.87 �^ 0.29 1.48 �^ 0.31 0.57 �̂ 0.28 ( p , 0.02)

*p-value compared with FALS normal diet.

Fig. 6 Glutamate concentrations in micro-

dialysates from cerebral cortex of G93A

transgenic mice and littermate controls fol-

lowing the administration of NMDA. NMDA

induced signi®cantly greater increases in

microdialysate glutamate concentrations in

G93A mice, as compared with littermate

controls (*p , 0.05). X, SOD1; W, wild

type.

Fig. 7 Glutamate concentrations in micro-

dialysates from cerebral cortex of G93A

transgenic mice and littermate controls at

baseline and following K1-induced gluta-

mate release. There were no signi®cant dif-

ferences. X, SOD1; W, wild type.

Creatine effects on glutamate in ALS mice 387

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390

the G93A mice did not signi®cantly differ from littermate

control mice (Fig. 7). Infusion of LTPD for 40 min signi®-

cantly increased the glutamate levels, and the G93A mice

showed signi®cantly higher increases than wild-type con-

trols at the later time points after the stimulation (Fig. 8,

p , 0.001 anova). The baseline levels of glutamate in all

the G93A mice used in the three experiments (n � 27) were

not different from the levels of the wild-type mice (n � 34).

The baseline concentration of glutamate in cortex was

0.50 ^ 0.07 mm in the transgenic G93A mice as compared

with 0.48 ^ 0.04 mm in wild-type littermates.

Discussion

In the present study using in vivo microdialysis we found

that NMDA-stimulated and LTPD-induced increases in

extracellular glutamate concentrations in microdialysates

were signi®cantly greater in G93A transgenic ALS mice

than in age-matched littermate controls. Concentrations of

glutamate in cortical tissue were also increased as measured

with NMR spectroscopy. Creatine supplementation attenu-

ated the increases in glutamate at 80, but not at 110 days

of age.

Previous studies using transgenic ALS mice showed

decreased glutamate transport in synaptosomes of spinal

cord from 150-day-old G93A mice (Canton et al. 1998), and

in cortex of 90-day-old G93A mice (Guo et al. 2000). There

was also increased vulnerability to glutamate toxicity in

cultured motor neurons from G93A mice (Kruman et al.

1999), and in neurons in which superoxide dismutase (SOD)

mutations were expressed (Roy et al. 1998). There is a 50%

reduction in EAAT-2 immunoreactivity at end stage illness

in G85R transgenic ALS mice (Bruijn et al. 1997). A recent

study showed elevated baseline glutamate and aspartate

concentrations, as well as reduced glutamate extraction, in

response to a challenge in end stage ALS mice as assessed

by in vivo microdialysis (Alexander et al. 2000).

In the present study we showed increased glutamate

concentrations in the cerebral cortex of ALS mice as

assessed by both microdialysis and magnetic resonance

imaging spectroscopy. The larger increase in glutamate

concentration induced by the glutamate reuptake inhibitor

LTPD in the ALS mice may be caused by a defect in

glutamate transport. If there is a reduced number or

impaired function of the transporters, a reduced capacity

for glutamate uptake may make them more susceptible to

additional inhibition. The larger increase in glutamate in

the ALS mice, as compared with controls, after NMDA

infusion may also be caused by a dysfunctional glutamate

re-uptake system.

The increase in both extracellular glutamate and the

total glutamate metabolite pool as measured with NMR

also ®ts with a reduced glutamate transport capacity.

Interestingly, the glutamate concentrations as assessed by

NMR were increased at 80 days of age preceding both the

onset of motor weakness and loss of motor neurons. There

are, however, other potential explanations. The glutamate±

glutamine cycling in the glia and neurons is a complex

process, where the enzyme glutamine synthetase plays a

vital role. There may be a speci®c impairment of glutamine

synthetase, which is very susceptible to oxidative damage,

leading to a decreased metabolism of glutamate to glutamine

(Oliver et al. 1990). An increase in tissue levels of

glutamate/glutamine could also re¯ect impaired energy

metabolism, similar to observations in Huntington's disease

transgenic mice (Jenkins et al. 2000).

It is known that the glutamate transporters are very

susceptible to oxidative stress, which may account for the

observed increases in glutamate concentrations (Volterra

et al. 1994). There is substantial evidence for oxidative

damage in transgenic mice with the G93A SOD mutations

(Ferrante et al. 1997; Andrus et al. 1998; Liu et al. 1999).

Using microdialysis in vivo we found evidence for increased

generation of hydroxyl radicals, as did others (Bogdanov

Fig. 8 Glutamate concentrations in micro-

dialysis samples from cerebral cortex of

G93A transgenic mice and littermate con-

trols following perfusion with the glutamate

reuptake inhibitor L-trans-pyrrolidine-2,4-

dicarboxylate (LTPD). The glutamate con-

centrations were signi®cantly increased in

the G93A transgenic mice, as compared

with wild-type controls (*p , 0.05). X, SOD1;

W, wild type.

388 O. A. Andreassen et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 383±390

et al. 1998; Liu et al. 1999). Increased free radical

generation was also shown with spin-trapping compounds

in vivo (Lin et al. 1998). There is also evidence for

increased 3-nitrotyrosine levels, a marker of peroxynitrite

mediated damage, in the ALS transgenic mice (Bruijn et al.

1997; Ferrante et al. 1997). Both oxidative damage and

nitration of the glutamate transporter leads to its inactivation

(Volterra et al. 1994; Trotti et al. 1996). Lipid peroxidation,

which occurs in transgenic ALS mice, can also impair

glutamate transport (Keller et al. 1997).

Recent studies showed that oxidative reactions triggered

by hydrogen peroxide were catalyzed by the A4V and I113T

SOD1 mutations, and that this led to inactivation of the

human EEAT2 glutamate transporter (Trotti et al. 1999).

The transporter was oxidized in its intracellular carboxyl-

terminal domain, and antioxidants prevented its inactivation.

It is therefore plausible that the increases in extracellular

glutamate we observed are a result of oxidative damage to

glutamate transporters.

Creatine (2%) signi®cantly improved motor performance

and increased longevity in the FALS mice, in accordance

with our previous ®ndings (Klivenyi et al. 1999). We also

examined whether 3% creatine would exert any further

bene®t on survival over 2% creatine. In accordance with our

prior study, 2% creatine produced better survival than 1%

creatine (Klivenyi et al. 1999). Supplementation with 3%

creatine, however, produced no additional bene®t and it was

equivalent to 1% creatine consistent with a U-shaped dose±

response curve. These results are consistent with our recent

results in a transgenic mouse model of Huntington's disease

(Ferrante et al. 2000). The explanation for this decreased

ef®cacy at higher concentrations is unclear.

Creatine can exert several potential neuroprotective

effects, including buffering of intracellular energy reserves,

stabilizing intracellular calcium concentrations and inhibit-

ing activation of the mitochondrial permeability transition

pore (O'Gorman et al. 1996; Matthews et al. 1998). By

increasing energy production, creatine may have a bene®cial

effect in removing glutamate from the synapse, which is a

very energy demanding process. Phosphocreatine can serve

as a direct energy source to stimulate synaptic glutamate

uptake and thereby reduce extracellular glutamate (Xu et al.

1996). The present results showing that creatine supplemen-

tation can reduce cortical glutamate concentrations are

consistent with this supposition. It is, however, also possible

that the reduction in cellular glutamate/glutamine concen-

trations at 80 days of age may re¯ect the ability of creatine

to simulate brain metabolism (Saks et al. 2000).

In conclusion, the present results show that transgenic

ALS mice have higher concentrations of glutamate in the

cerebral cortex as measured by microdialysis and NMR

spectroscopy. This is consistent with a role of increased

glutamate concentrations in the disease process. Creatine

supplementation reduces the increased glutamate levels,

which may contribute to bene®cial effects of creatine on

longevity and motor performance in these mice.

Acknowledgements

The secretarial assistance of Sharon Melanson is gratefully

acknowledged. This work was supported by NIA grant P01

AG12992, and the ALS Association. OAA is supported by the

Norwegian Research Council.

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