expression of groups i and ii metabotropic glutamate receptors in the rat brain during aging

www.elsevier.com/locate/brainres

Brain Research 1043

Research report

Expression of groups I and II metabotropic glutamate receptors in the

rat brain during aging

Agnes Simonyia,T, Richard T. Ngombab, Marianna Stortob, Maria V. Cataniac, Laura A. Millera,

Brian Youngsa, Valeria DiGiorgi-Gerevinid, Ferdinando Nicolettib,d, Grace Y. Suna

aDepartment of Biochemistry, University of Missouri, M743 Medical Sciences Bldg., Columbia, MO 65212, USAbI.N.M. Neuromed, Loc. Camerelle, 86077 (Isernia), Pozzilli, Italy

cCNR, Inst. Neurol. Sci., Viale Regina Margherita 6, I-95123 Catania, ItalydDepartment of Human Physiology and Pharmacology, University of Rome, dLa SapienzaT, Piazzale Aldo Moro 5, I-00185 Rome, Italy

Accepted 15 February 2005

Available online 24 March 2005

Abstract

Age-dependent changes in the expression of group I and II metabotropic glutamate (mGlu) receptors were studied by in situ hybridization,

Western blot analysis and immunohistochemistry. Male Fisher 344 rats of three ages (3, 12 and 25 months) were tested. Age-related increases

in mGlu1 receptor mRNA levels were found in several areas (thalamic nuclei, hippocampal CA3) with parallel increases in mGlu1a receptor

protein expression. However, a slight decrease in mGlu1a receptor mRNA expression in individual Purkinje neurons and a decline in

cerebellar mGlu1a receptor protein levels were detected in aged animals. In contrast, mGlu1b receptor mRNA levels increased in the

cerebellar granule cell layer. Although mGlu5 receptor mRNA expression decreased in many regions, its protein expression remained

unchanged during aging. Compared to the small changes in mGlu2 receptor mRNA levels, mGlu3 receptor mRNA levels showed substantial

age differences. An increased mGlu2/3 receptor protein expression was found in the frontal cortex, thalamus, hippocampus and corpus

callosum in aged animals. These results demonstrate region- and subtype-specific, including splice variant specific changes in the expression

of mGlu receptors in the brain with increasing age.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Excitatory amino acid receptors: structure, function and expression

Keywords: Aging, metabotropic glutamate receptors; Rat brain; In situ hybridization; Western blot; Immunohistochemistry

1. Introduction

Normal aging is accompanied by alterations of many

neurotransmitter and second messenger systems in the brain.

Recently, the glutamatergic neurotransmitter system has

received a great deal of attention in particular, in research

examining impairments in normal and pathological aging.

Several studies have recognized age-related changes in the

density and function of the different ionotropic glutamate

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.02.046

T Corresponding author. Fax: +1 573 884 4597.

E-mail address: [email protected] (A. Simonyi).

receptors [22,26,29,57]. Although metabotropic glutamate

(mGlu) receptors are involved in synaptic plasticity [2],

there are only few studies that have investigated age-related

changes in the characteristics of mGlu receptor neuro-

transmission due to lack of subtype-specific agonists and/or

antagonists. Metabotropic glutamate receptors form a family

of at least eight subtypes [11], which are subdivided into

three groups on the basis of sequence homology, pharma-

cological profile of activation, transduction pathways and

several of these have multiple splice variants. While group I

mGlu receptors (mGlu1a, b, c, d, g and mGlu5a, b) are

coupled to the polyphosphoinositide signaling pathway,

group II mGlu receptors (mGlu2 and mGlu3) and group III

(2005) 95–106

Fig. 1. Autoradiograms showing group I (mGlu1 and mGlu5) and II (mGlu2 and mGlu3) mGlu receptor mRNA expression in coronal sections of rat brain at

3 months of age.

Fig. 2. (A) Representative immunoblot of mGlu1a receptors in the striatum

of rats at 3, 12 and 25 months of age. (B) Changes in the expression of

mGlu1a receptor proteins in the cerebral cortex (CTX), corpus striatum

(CS) and cerebellum (CER) of aged rats, as assessed by Western blot

analysis. Data are expressed as percent of the corresponding values found in

rats at 3 months of age, and were calculated by densitometric analysis as the

ration between the mGlu1a receptor and h-actin. Data are meansF SEM of

4–6 determinations. *P b 0.05 (Student’s t test), as compared with the

corresponding values at 3 months.

A. Simonyi et al. / Brain Research 1043 (2005) 95–10696

mGlu receptors (mGlu4a, b; mGlu6; mGlu7a, b; and

mGlu8a, b) are linked to the inhibition of cAMP cascade

[11,36].

An early study by Parent et al. [33] investigated the group

I/II mGlu receptor agonist trans-1-amino-cyclopentyl-1,3-

dicarboxylate (ACPD) stimulated inositol phosphate (IP)

turnover in aged Long–Evans rats based on their performance

in the Morris water maze. They found an increase in IP

turnover in the frontal cortex and the hippocampus of

cognitive impaired animals as compared to the aged unim-

paired and the young rats. In addition, a significant decrease

in IP turnover was shown in the parietal cortex which was

independent of the cognitive performance. In contrast,

Nicolle et al. [30] showed a decreased IP turnover in the

hippocampus of aged rats, which was correlated with the

impairment in the water maze learning. Receptor binding

studies with C57B1 mice did not find correlation with

performance in the Morris water maze and the results

revealed no changes in receptor binding during aging, but

the metabotropic type 1 (high quisqualate affinity) binding

sites showed a declining trend, especially in the hippocampus

[21]. McGahon and Lynch [25] showed an age-related deficit

in the glutamate release by adding ACPD and arachidonic

acid to hippocampal synaptosomes. Another pertinent find-

ing is that ACPD increased dopamine efflux in the prefrontal

cortex of aged rats but had no effect in young rats [38].

These studies suggested further investigations to deter-

mine the effect of aging on specific subtypes of mGlu

receptors. Our earlier study showed a significant decrease

A. Simonyi et al. / Brain Research 1043 (2005) 95–106 97

in the level of mGlu1a receptor protein in the cerebellum

of 24-month-old C57BL/6NNIA mice as compared to the

5- and 15-month-old groups. However, a progressive

increase in the mRNA level of mGlu1 receptor was found

in the granule cell layer during aging [49]. In the present

study, we systematically analyzed region- and subtype-

specific, including splice variant-specific changes in the

expression of group I and group II mGlu receptors in

Fisher 344 rat brain during aging by quantitative in situ

hybridization, Western blot analysis and immunohisto-

chemistry.

2. Materials and methods

2.1. Animals and tissue preparations

Male Fisher 344 rats of three age groups were used, 3-

month-old (young adult), 12-month-old (middle-aged) and

25-month-old (aged). All animals were obtained from the

National Institute on Aging colonies (Harlan, Indianapolis,

IN). Animals were acclimatized for a week before use. On

the day of the experiment, animals were anesthetized with

isoflurane and decapitated. The brains were removed and

frozen in powdered dry ice.

Fig. 3. Age-dependent changes in the expression of mGlu1a receptor protein in dif

are means F SEM of 4–5 determinations. *P b 0.05 (One-way ANOVA + Fishe

2.2. In situ hybridization

Twelve Am coronal or sagittal sections were used for

in situ hybridization. Sections were fixed in 4%

paraformaldehyde/phosphate buffered saline (PBS) for 5

min, rinsed in PBS for 2 min, and soaked in 0.25%

acetic anhydride in 0.1 M triethanolamine hydrochloride/

0.9% NaCl (pH 8.0) for 10 min. They were rinsed in

2� SSC (300 mM NaCl/30 mM sodium citrate),

dehydrated through a graded series of ethanol, delipi-

dated in chloroform, rehydrated to 95% ethanol and air-

dried. Fifty Al of hybridization buffer was applied to

each slide, covered with a parafilm coverslip and

incubated at 42 8C overnight. The hybridization buffer

contained 50% formamide, 4 � SSC, transfer RNA (250

Ag/ml), sheared, single-stranded salmon sperm DNA (100

Ag/ml), 1� Denhardt’s solution (0.02% each of BSA,

Ficoll and polyvinylpyrrolidone), 10% (w/v) dextran

sulfate (MW 500,000), 200 mM DTT and 0.75 � 106

cpm probe. After hybridization, coverslips were removed

in 1� SSC. Slides were washed in 1 � SSC (2 mM

DTT) at 55 8C for 4 � 15 min. Following two 30-min

rinses in 1 � SSC at room temperature, the tissues were

dipped in distilled water, immersed in 70% ethanol and

air-dried.

ferent regions of the rat brain, as assessed by immunohistochemistry. Values

r’s PLSD) vs. the corresponding values at 3 months.

Fig. 4. Representative images of mGlu1a (A–B) and mGlu2/3 (C–D)

immunolabeled sections of 3-month-old (A and C) and 25-month-old

animals (B and D).


2.3. Probes

Oligonucleotides were 3Vend-labeled by terminal deoxy-

nucleotidyl transferase (Boehringer-Mannheim, Indianapo-

lis, IN) with 35S-dATP (NEN, Boston, MA). The probes for

mGlu1 and mGlu5 receptors were the same as used earlier

[50]. The following sequences were used for the mGlu1

receptor splice variant-specific probes: mGlu1a, 45-mer

oligonucleotide complementary to bases 2642–2686 [54];

mGlu1b, 45-mer oligonucleotide complementary to bases

Table 1

Age-dependent changes in the mRNA expression of mGluR1 in rat brain

Regions 3-month-

old

12-month-

old

25-month-

old

P values

Caudate putamen 63 F 1.0 57 F 2.5 59 F 1.8 0.0966

Frontal cortex 49 F 1.3 48 F 2.3 51 F 1.0 0.4330

Parietal cortex 54 F 1.4a 54 F 1.7 64 F 2.5b 0.0019

Piriform cortex 99 F 3.3 96 F 2.1 101 F 3.2 0.4893

CA1 43 F 3.0 44 F 3.7 44 F 2.5 0.9661

CA3 148 F 5.0a 142 F 3.8 163 F 5.1b 0.0149

Dentate gyrus—

upper blade

165 F 5.9 173 F 4.2 171 F 4.0 0.4822

Dentate gyrus—

lower blade

194 F 10.0 199 F 7.0 205 F 6.9 0.6368

Laterodorsal

thalamic nu

133 F 2.2a 143 F 4.2c 180 F 2.8b b0.0001

Ventral posterol.—

m. thal nu

69 F 1.2a 90 F 2.3c 119 F 7.6b b0.0001

Temporal cortex 28 F 1.8 27 F 1.0 30 F 1.2 0.3151

Occipital cortex 55 F 0.6a 57 F 1.1 59 F 0.5 0.0071

Entorhinal cortex 15 F 0.8 13 F 0.9 13 F 0.7 0.1562

Data are expressed in nCi/g tissue F SEM from 7 animals/group.

One-way ANOVA, Newman–Keuls multiple comparison test.a Significant difference (P b 0.05 or lower) between 3-month-old and 25-

month-old.b Significant difference (P b 0.05 or lower) between 12-month-old and 25-

month-old.c Significant difference (P b 0.05 or lower) between 3-month-old and 12-

month-old.

2656–2700 [54]; mGlu1c, a mixture of oligonucleotides

complementary to bases 2688–2720 and 2658–2693 [37];

mGlu1d, 45-mer oligonucleotide complementary to bases

2653–2697 [23]. The mGlu2 receptor probe corresponded to

nucleotides 367–411 of rat brain cDNA [54]. The probe for

mGlu3 receptor corresponded to nucleotides 2521–2565 of

rat brain cDNA [54].

2.4. Autoradiography and signal quantitation

Slides were held against KODAK BIOMAX MR films

with standards (American Radiolabeled Chemicals, St.

Louis, MO) in X-ray cassettes. Microdensitometry was

performed on the signal over different brain regions using

the BIOQUANT True Color Windows 95 software version

2.50 as earlier described [51]. [14C]-microscale standards

were used to construct calibration curves and quantitate

signals. mRNA levels (nCi/g tissue) were averaged from the

analysis of four sections for each animal (six-seven per

group) before being evaluated for statistical significance

(One-way ANOVA, Newman–Keuls multiple comparison).

Fig. 5. Representative photomicrographs of the hippocampal CA3 neurons

illustrating emulsion autoradiograms of in situ hybridization with 35S-labeled

probe for mGlu1 receptor mRNA. Top: 3-month-old; Bottom: 25-month-old.

Magnification 100�.


Sections were used at three coronal levels (Fig. 1). The

Paxinos–Watson atlas [34] was used for identification of

brain nuclei. Emulsion autoradiography using sagittal

sections of the cerebellum and signal quantitation were

carried out as described earlier [48,51].

2.5. Western blot analysis

The expression of mGlu receptor proteins was estimated

by Western blot analysis, using antibodies raised against

synthetic peptides corresponding to the following carboxy-

terminal amino acid sequences (one-letter code): NGRE-

VVDSTTSSL (13 carboxy-terminal residues of mGlu

receptor; commercially available, Chemicon International,

Temecula, CA) to label mGlu2 or 3 receptors, KSPKYDT-

LIIRDYTNSSSSL (21 carboxy-terminal residues of mGlu5

receptor) to label both mGlu5a and -5b receptors and

KPNVTYASVILRDYKQSSSTL (21 carboxy-terminal res-

idues of mGlu1a receptor) to label mGlu1a receptors

(commercially available, Upstate Biotechnology, Lake

Placid, NY), and a monoclonal antibody to label h-actin(commercially available, Sigma, St. Louis, MO). Tissues

were homogenized at 4 8C in Tris–HCl buffer (20 mM, pH

Fig. 6. Age-dependent changes in the mRNA expression of mGlu1 receptor sp

animals/groups. mGluR1 granule cell layer—P b 0.0001. (a) P b 0.001 compa

Purkinje cells—P = 0.0406. mGluR1b granule cell layer—P = 0.0149. (a) P

multiple comparison test.

7.4 containing 10% sucrose). Homogenates were sequen-

tially centrifuged at 1500 � g for 20 min and the resulting

supernatant was centrifuged at 20,000 � g to obtain the P2

fraction. Pellets were resuspended in ice-cold Tris–HCL

buffer containing 1 mM PMSF, pH 7.4, and an aliquot was

used for protein determinations. Proteins were resuspended

in SDS-bromophenol blue reducing buffer with 40 mM

DTT to limit the formation of high molecular weight

receptor aggregates. Comassie-stained SDS polyacrylamide

gels were run on a minigel apparatus (BIORAD Mini

Protean II cell); gels were electroblotted on Immuno PVDF

membrane (Biorad, Italy) for 1 h using a semi-dry

electroblotting system (BIORAD, Trans-blot system SD),

and filters were blocked overnight in TTBS (100 mM Tris–

HCL; 0.9% NaCl, 1% Tween 20, pH 7.4) containing 2%

non-fat dry milk. Blots were then incubated for 1 h at room

temperature with primary polyclonal antibodies, (concen-

tration: 1 Ag/ml for mGlu5 and -1 receptors, 0.5 Ag/ml for

mGlu2/3 receptors and 1.3 Ag/ml for h-actin). Blots were

washed three times with TTBS buffer and then incubated

for 1 h with secondary antibodies (peroxidase-coupled anti-

rabbit, Amersham Pharmacia Biotech, Arlington Height,

IL) diluted (1:10,000) with TTBS. Immunostaining was

lice variants in the cerebellum. Values are means F SEM from 6 to 7

red to 25-month-old; (b) P b 0.001 compared to 3-month-old. mGluR1a

b 0.05 compared to 25-month-old. One-way ANOVA, Newman–Keuls

Fig. 7. (A) Representative immunoblot of mGlu2/3 receptors in the

hippocampus and cerebral cortex of rats at 3, 12 and 25 months of age.

Bands corresponding to monomeric and dimeric mGlu2/3 receptors are

shown. (B) Changes in the expression of mGlu2/3 receptor proteins in the

hippocampus (Hippo), cerebral cortex (CTX), cerebellum (CER) and

corpus striatum (CS) of aged rats, as assessed by Western blot analysis.

Data are expressed as percent of the corresponding values found in rats at

3 months of age, and were calculated by densitometric analysis as the

ration between the mGlu2/3 receptor and h-actin. Data are means F SEM

of 3–5 determinations. *P b 0.05 (Student’s t test), as compared with the

corresponding values at 3 months.


revealed by the enhanced ECL Western Blotting analysis

system (Amersham Pharmacia Biotech, Arlington Height,

IL). The intensity of the bands was quantitated by image

analysis. When two bands were present (receptor mono-

mers and dimers), the sum of the two bands was

normalized by the levels of h-actin.

2.6. Immunohistochemistry and signal quantitation

Immunohistochemistry and signal quantitation were

carried out as described previously [10]. Briefly, animals

were perfused with 0.9% NaCl solution followed by 4%

paraformaldehyde in PBS. Brains were removed, postfixed

overnight (o.n.) and immersed in a sterile cryoprotective

solution of 30% sucrose at 4 8C. Forty-micrometer-thick

sections were cut on a cryotome and immediately

processed for immunohistochemistry. Sections were trans-

ferred into 50 mM Tris–HCl buffer containing 1.5% NaCl,

pH 7.4 (TBS) and then permeabilized for 30 min in TBS

with 0.4% Triton X-100. This was followed by a

preincubation in TBS containing 4% normal goat serum

(NGS). Sections were then incubated o.n. in primary

antibodies in TBS containing 0.1% Triton X-100,2%

NGS. Rabbit polyclonal anti-mGluR1 (1:1000; Upstate

Biotechnology, Lake Placid, NY), anti-mGluR2/3 (1:200;

Chemicon International, Temecula, CA) and anti-mGluR5

(1:200; Upstate Biotechnology, Lake Placid, NY) were

used. On the following day, sections were washed with

cold TBS and incubated for 2.5 h in biotinylated goat

anti-rabbit antibodies (1:200, Vector Laboratories, Burlin-

game, CA). Sections were extensively rinsed in TBS and

then incubated for 45 min in the ABC Elite reagent

(Vector Laboratories, Burlingame, CA). Color develop-

ment was achieved by incubating the slices in a Tris–HCl

(50 mM) solution containing 3,3V-diaminobenzidine (final

concentration 375 Ag/ml) and H2O2 (final concentration

0.0045%). Sections were rinsed in TBS, mounted on

gelatin-coated slides, dehydrated in increasing concentra-

tions of ethanol, clarified and coverslipped in a xylene-

based mounting medium. Sections from different animals

were processed in parallel and the time of 3,3V-diamino-

benzidine/H2O2 development was identical for each

section. Signal was quantified by computer-assisted

densitometry, using the MCID system (Imaging Research,

St. Catharine’s, Ontario, Canada). Images were visualized

under the same light conditions on a video monitor

connected to the microscope through a video camera. The

integrated optical density (OD) was obtained by the

software operated conversion of absolute gray values in

arbitrary OD units. This computation was done after

obtaining a linear calibration curve generated by the

system, attributing the arbitrary value of 0 to the lightest

gray value and 3 to the highest value. These values were

averaged from several readings in different sections. Data

were analyzed by one-way ANOVA followed by Fisher’s

PLSD.

3. Results

3.1. Age-dependent changes in the expression of mGlu1

receptors

3.1.1. Changes in mGlu1a receptor protein

Western blot analysis of mGlu1a receptors revealed a

major band at 145 kDa corresponding to receptor mono-

mers. Consistent with previous findings [49], mGlu1a

receptor expression was reduced in the cerebellum of 25-

month-old rats. Interestingly, we observed a substantial

increase in mGlu1a receptor expression in the caudate–

putamen of aged rats, while a smaller increase was observed

in the cerebral cortex of 12-month old rats (Fig. 2).

Immunohistochemical analysis of mGlu1a receptors con-

firmed the age-dependent increase in the striatum and

cerebral cortex. An increase was also observed in thalamic

nuclei and hippocampal CA1 and CA3 regions of aged rats

(Figs. 3 and 4).


3.1.2. Changes in mGlu1 receptor mRNA

Quantitative in situ hybridization revealed a substantial

increase in mGlu1 receptor mRNA levels in the thalamic

nuclei of aged rats, with a greater increase being

observed in 25-month-old rats (Table 1). Among the

cortical regions, a slight increase in the mGlu1 receptor

mRNA levels was observed in the parietal and occipital

cortices during aging. In the hippocampus, mGlu1

receptor mRNA level was elevated in the CA3 area at

25 months of age as compared to the two younger groups

(Table 1). Since recent studies demonstrated no changes

in cell numbers in the hippocampus during aging [42],

we examined the expression of mGlu1 receptor mRNA at

the cellular level by emulsion-coating the sections used

for film autoradiographs. Fig. 5 shows the increase in

mGlu1 receptor mRNA expression over individual cells

in the CA3 area. No age-dependent changes were

detected in other hippocampal subregions by in situ

hybridization.

In the cerebellum, we confirmed the age-dependent

increase in mGlu1 receptor mRNA levels in the granular

layer (with no change in the Purkinje cell layer) in spite of

the reduction of mGlu1a receptor protein observed by

Western blot analysis (see Fig. 2, and also Ref. [49]). This

prompted us to extend the in situ hybridization analysis to

individual splice variants of mGlu1 receptors. Interestingly,

mGlu1b mRNA levels were substantially increased in the

Fig. 8. Age-dependent changes in the expression of mGlu2/3 receptor protein in dif

are means F SEM of 4 determinations. *P b 0.05 (One-way ANOVA + Fisher’s

granular layer of 25-month-old rats, whereas mRNA levels

of mGlu1a, mGlu1c (data not shown) and mGlu1d receptors

remained unchanged during aging (Fig. 6). Cellular

quantitation of the splice variants’ mRNAs in the Purkinje

neurons revealed a significant linear trend to decrease for

the major form mGlu1a receptor. This decrease reached

about 20% in aged animals showing a typical high variation

in this group (Fig. 6).

3.2. Age-dependent changes in the expression of mGlu2 and

-3 receptors

3.2.1. Changes in mGlu2/3 receptor proteins

Western blot analysis of mGlu2/3 receptors consistently

showed a doublet at 100 kDa, which may represent

receptor monomers, and a higher molecular weight band,

which represents receptor dimers [40]. Expression of

mGlu2/3 receptors showed an age-dependent increase in

all the brain regions that we have selected for Western blot

analysis, that is, striatum, cerebral cortex, cerebellum and

hippocampus. In all regions, the increase was more

remarkable at 25 than at 12 months (Fig. 7). These results

were roughly confirmed by immunohistochemical analysis,

which showed a remarkable increase in mGlu2/3 receptor

expression in the striatum, frontal cortex, thalamic nuclei

and hippocampal subfields. However, there was a discrep-

ancy in the cerebellum, in which no increases were


PLSD) vs. the corresponding values at 3 months.

Table 2


Regions 3-month-

old

12-month-

old

25-month-

old

P values

Caudate putamen 16 F 1.0a 20 F 1.0b 20 F 0.8 0.0097


Parietal cortex 48 F 1.2 49 F 1.5 45 F 1.8 0.1820

Dentate gyrus—

upper blade

476 F 11a 415 F 10b 427 F 7.0 0.0006

Dentate gyrus—

lower blade

424 F 16 391 F 14 405 F 12 0.2767


Occipital cortex 186 F 1.5 188 F 3.3 190 F 3.2 0.6355


Cerebellar granule

cell layer

25 F 3.3 26 F 2.3 27 F 1.9 0.8604

Data are expressed in nCi/g tissue F SEM from 6 to 7 animals/group.

One-way ANOVA, Newman–Keuls multiple comparison test.a Significant difference (P b 0.05 or lower) between 3-month-old and

25-month-old.b Significant difference (P b 0.05 or lower) between 3-month-old and

12-month-old.


detected by immunohistochemistry. This is difficult to

explain because it is in the cerebellum that we have

detected the greater age-dependent increase in the expres-

sion of mGlu2/3 receptors by Western blot analysis. It is

possible that most of the protein(s) detected by immuno-

Table 3


Regions 3-month-

old

12-month-

old

25-month-

old

P values

Caudate putamen 106 F 4.9 109 F 4.4 120 F 3.8 0.0861

Nucleus accumbens—

core

118 F 5.6a 122 F 2.0 136 F 5.3 0.0371


shell

103 F 9.3 111 F 5.4 120 F 3.9 0.2172



Dentate gyrus—

upper blade

199 F 5.5 210 F 6.6 210 F 7.1 0.3966

Dentate gyrus—

lower blade

175 F 4.4a 187 F 5.8 203 F 6.8 0.0103

Reticular thalamic

nucleus

189 F 9.1a 217 F 6.5b 260 F 7.3c b0.0001


Occipital cortex 115 F 2.4a 124 F 2.4b 137 F 2.7c b0.0001


Central gray 53 F 1.1a 81 F 4.2b 124 F 4.2c b0.0001

Corpus callosum 196 F 8.3a 260 F 9.9b 333 F 7.9c b0.0001

Internal capsule 147 F 9.6a 134 F 7.9 196 F 17c 0.0050

Cerebellar granule

cell layer

74 F 3.1a 74 F 1.6 86 F 2.8c 0.0051

Data are expressed in nCi/g tissue F SEM from 6 to 7 animals/group.



12-month-old.c Significant difference (P b 0.05 or lower) between 12-month-old and

25-month-old.

blotting in the cerebellum is not expressed at the cell

surface, and, therefore cannot be detected by immunohis-

tochemical analysis. Interestingly, a substantial increase in

the expression of mGlu2/3 receptor protein was observed

in the corpus callosum of 25-month-old rats (Figs. 4 and

8).

3.2.2. Changes in mGlu2 and -3 receptor mRNA

In situ hybridization analysis showed that mGlu2

receptor mRNA levels remained stable in most areas

examined during aging. A slight increase in mGlu2

receptor mRNA was observed in the caudate–putamen

of 12- and 25-month-old animals, whereas a reduction

was observed in the upper blade of the dentate gyrus

(Table 2). In contrast, mGlu3 receptor mRNA levels

underwent major changes during aging. Similarly to

immunohistochemical data, a substantial increase in

mGlu3 receptor mRNA levels was observed in the corpus

callosum and internal capsule (i.e., in the white matter) of

aged rats. Large increases in mGlu3 receptor mRNA

levels during aging were also observed in the reticular

thalamic nucleus and central gray, whereas smaller

changes were present in the occipital cortex, outer blade

of the dentate gyrus, nucleus accumbens and cerebellar

granule cell layer (Table 3).

Fig. 9. (A) Representative immunoblot of mGlu5 receptors in the

cerebellum and cerebral cortex of rats at 3, 12 and 25 months of age.

Bands corresponding to monomeric and dimeric receptors and h-actin are

shown. (B) Changes in the expression of mGlu5 receptor proteins in the

cerebellum (CER) and cerebral cortex (CTX) of aged rats, as assessed by

Western blot analysis. Data are expressed as percent of the corresponding

values found in rats at 3 months of age, and were calculated by

densitometric analysis as the ration between the mGlu5 receptor and h-actin. Data are meansF SEM of 4–5 determinations. *P b 0.05 (Student’s t

test), as compared with the corresponding values at 3 months.


3.3. Age-dependent changes in mGlu5 receptors

3.3.1. Changes in mGlu5 receptor proteins

Western blot analysis of mGlu5 receptors showed a major

band at 145 kDa corresponding to receptor monomers, and a

light band of higher molecular weight, corresponding to

receptor dimers. Both bands were absent in brain tissue of

mGlu5 knockout mice [5]. A trend to a reduction in the

expression of mGlu5 receptors was observed in the striatum,

hippocampus and cerebral cortex of 25-month-old rats. These

changes, however, were not statistically significant. In

contrast, a substantial increase in mGlu5 receptor protein

was observed in the cerebellum of 25-month-old rats. In 12-

month-old rats, the only significant change we have observed

by Western blot analysis was a slight increase in the cerebral

cortex (Fig. 9). Immunohistochemical analysis did not show

any significant changes in the expression of mGlu5 receptors

across the brain of aged rats (Fig. 10).

3.3.2. Changes in mGlu5 receptor mRNA

In situ hybridization analysis showed a reduction in

mGlu5 receptor mRNA levels in the caudate–putamen,

occipital, temporal and entorhinal cortices, hippocampal

CA3 region, nucleus accumbens (core), and central gray of

aged rats. Interestingly, age-dependent changes in the

expression of mGlu5 receptor mRNA showed an opposite

trend in the cerebellum. A significant increase in mRNA

Fig. 10. Age-dependent changes in the expression of mGlu5 receptor protein in dif

are means F SEM of 4–5 determinations.

level was found in the granule cell layer at 25 months of age

(Table 4).

4. Discussion

4.1. Age-associated changes in group I mGlu receptors

There are only few reports on the effect of aging on the

expression and function of group I mGlu receptors. A recent

study showed a decrease in the stimulation of PI hydrolysis

by mGlu receptor agonists in the hippocampus of aged

animals, which was significantly correlated with the impair-

ment of spatial learning [30]. This effect, however, reflected

a reduction in phospholipase-Ch1 levels, rather than

changes in the expression of mGlu1 or -5 receptors [30].

The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine

(DHPG), enhanced striatal dopamine release in young

animals, but produced opposite effects in aged animals

[39]. Age-related changes in mGlu receptor binding were

reported in C57B1 mice. Binding to the so-called met-1

sites, which should incorporate both mGlu1 and -5

receptors, does not change substantially in the brain of

aged animals, with the exception of a decrease in the CA1

stratum lacunosum/moleculare and in the lower blade of the

dentate gyrus [21]. However, no systematic studies have

been carried out on how expression of individual mGlu


Table 4


Regions 3-month-

old

12-month-

old

25-month-

old

P values

Caudate putamen 113 F 2.9a 110 F 2.9 96 F 5.0b 0.0107


core

107 F 6.0a 93 F 2.0c 88 F 5.0 0.0264


shell

98 F 5.8 86 F 4.2 88 F 5.4 0.2302



Piriform cortex 167 F 4.5 167 F 4.2 154 F 7.4 0.1900

CA1 200 F 9.4 197 F 4.0 190 F 8.0 0.6329

CA3 140 F 4.2a 118 F 4.1c 119 F 3.4 0.0012

Dentate gyrus—

upper blade

146 F 8.0 152 F 2.7 156 F 6.4 0.5206

Dentate gyrus—

lower blade

165 F 4.4 169 F 2.7 175 F 2.9 0.1434

Laterodorsal

thalamic nu

55 F 1.4 52 F 1.1 51 F 1.3 0.0961

Ventral posterol.—

m. thal nu

21 F 1.3 18 F 0.6 18 F 0.9 0.0673

Temporal cortex 32 F 1.5a 25 F 1.4c 24 F 1.1 0.0009

Occipital cortex 28 F 1.2a 23 F 0.7c 22 F 1.0 0.0009

Entorhinal cortex 37 F 2.5a 31 F 2.1 25 F 3.4 0.0205

Central gray 12 F 0.6a 8.4 F 0.9c 9.2 F 0.7 0.0077

Cerebellar granule

cell layer

66 F 3.8a 66 F 3.1 81 F 1.9b 0.0030

Data are expressed in nCi/g tissue F SEM from 7 animals/group.



25-month-old.c Significant difference (P b 0.05 or lower) between 3-month-old and

12-month-old.


receptor subtypes changes during aging. Here, we have

found that mGlu1 receptor mRNA levels (which represent

an overall estimate of all mGlu1 receptor splice variants)

were uniformly increased in the brain of aged rats.

Expression of the mGlu1a receptor protein was also

elevated in the striatum, cerebral cortex, CA1 and CA3

hippocampal subfields and thalamic nuclei of aged rats

suggesting that, at least in these regions, an increased de

novo synthesis of mGlu1a receptors occurs during aging. As

thalamic mGlu1 receptors have been implicated in noci-

ceptive processing [27,56], it will be interesting to examine

whether aged animals show changes in pain threshold, and

whether these changes are sensitive to mGlu1 receptor

antagonists. A different scenario was observed in the

cerebellum of aged animals, where the expression of

mGlu1a receptor protein was reduced in spite of an increase

in mGlu1 receptor mRNA levels (see also Ref. [49] for

similar results obtained in aged mice). Because of this

apparent discrepancy, we decided to extend the analysis to

the mRNAs encoding for the various splice variants of

mGlu1 receptors. In situ hybridization analysis showed a

distribution pattern of the different mGlu1 receptor splice

forms similar to that reported by other investigators [6,18].

Recently, a new alternatively spliced form, named mGluR1f,

was found [52]. Since the 85-bp insertion fragment of mGlu1f

is the same as in mGlu1b, our mGlu1b-specific probe was

also complementary to mGlu1f. We have shown that mGlu1b

(and/or mGlu1f) mRNA is increased in the granular layer at

25 months, when the pan-mGlu1 mRNA is also increased.

However, to what extent mGlu1b mRNA contributes to the

overall mGlu1mRNA is unclear because mGlu1b mRNA did

not increase at 12 months, in spite of the increase in pan-

mGlu1 mRNA. It will be interesting to examine whether the

mGlu1b/1f receptor protein also increases in the cerebellar

granule cell layer of aged rats. Among all the mGlu1 receptor

splice variants expressed in the cerebellum, only the func-

tional role of mGlu1a receptors in Purkinje cells is known at

present. Activation of mGlu1a receptors is necessary for the

induction of long-term depression at the synapses between

parallel fibers and Purkinje cells, which is a putative substrate

for motor learning [1,12]. In addition, production of anti-

mGlu1a receptor antibodies has been causally related to

paraneoplastic ataxia in patients with Hodgkin’s lymphoma

[47]. Hence, a reduction in mGlu1a receptors in Purkinje cells

may underlie the age-dependent impairment in motor

learning and motor coordination [43].

Age-dependent changes in mGlu5 receptors differed

from those exhibited by mGlu1 receptors. In most of the

forebrain regions, mGlu5 receptor mRNA levels were

reduced, and the expression of mGlu5 receptor protein

showed a clear (although not significant) trend to a

reduction in aged animals. Electron microscopic studies

show that mGlu5 receptors are predominantly localized in

postsynaptic densities, where they may be physically

coupled to NMDA receptors through a mechanism of

heterophylic protein-to-protein interaction [55]. Activation

of mGlu5 receptors positively modulates NMDA receptors

(see Ref. [9] and references therein) and appears to be

required for the induction of NMDA-dependent long-term

potentiation (LTP) and spatial learning [17,20]. Thus, a

reduced expression of mGlu5 receptors might contribute to

the attenuation of NMDA-mediated responses observed in

different brain regions of aged animals [4,7,16], and is in

line with the evidence that potentiation of NMDA responses

by DHPG is attenuated in the aged striatum [39].

4.2. Age-associated changes in group II mGlu receptors

Group II mGlu receptors (i.e., mGlu2 and -3 receptors)

are negatively coupled to adenylyl cyclase activity, although

they also regulate other signaling pathways, as well as a

variety of membrane ion channels (reviewed by Refs.

[11,44]). Interestingly, a reduction in adenylyl cyclase

activity and [3H] forskolin binding has been shown in

many brain regions in aged rats [3,31]. In this study, we

found an increase in the expression of group II mGlu

receptor proteins in most of the brain regions of aged rats.

This most likely reflected an increased de novo synthesis of

mGlu3 receptors, as inferred by measurements of mGlu2

and mGlu3 receptor mRNA levels. In neurons, mGlu2 and


-3 receptors are predominantly localized on presynaptic

terminals, where they negatively modulate glutamate

release [32,45,53]. Interestingly, the expression of mGlu7

receptors, which are also presynaptic and negatively

modulate glutamate release (reviewed by Ref. [13]), is

reduced during aging [51]. We speculate that the enhanced

expression of mGlu3 receptors represents a compensatory

mechanism aimed at limiting an excessive release of

glutamate during aging which was reported in several

brain regions including the striatum and the hippocampus

[15,24,41]. MGlu3 receptors are also present in glial cells

[35,53], and their expression increases in reactive astro-

cytes [14]. Although we could not identify the cellular

source of mRNA by film autoradiography, it is likely that

glial mGlu3 receptors largely contribute to the overall

increase in receptor expression observed in aged animals.

Accordingly, glial activation is an established feature of the

aging brain, and expression of glial fibrillary acidic protein

(GFAP) increases with age in the brain of rodents and

humans [19,28]. Using an intron-specific probe for GFAP

mRNA, Yoshida et al. [58] reported an almost 100%

increase in grain density in the corpus callosum and

internal capsule between 8 and 24 months in male Fisher

344 rats. Our study found a marked increase in mGlu3

receptor expression in the corpus callosum and white

matter of aged animals. The physiological role of mGlu3

receptors in glial cells has not yet been elucidated. At least

two functions have been ascribed to glial mGlu3 receptors:

(i) the regulation of water channel aquaporin 4, which is

involved in cell swelling [46]; and (ii) the production of

neurotrophic factors that protect neurons against excito-

toxic death [8]. Both aspects might be of great relevance

for pathological processes associated with aging.

Acknowledgments

This study was partially supported by NIH NIA 1P01

AG18357 and the Missouri’s Alzheimer’s Association.

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