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1 Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra 2 Laboratory of Neurosciences, Faculty of Medicine, University of Lisbon, 1649-028 Lisboa, Portugal
Submitted 7 October 2002; accepted in final form 7 December 2002
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ABSTRACT |
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–receptor agonist, isoproterenol (30 µM),
CGS21680 (100–1,000 nM) enhanced cAMP levels in hippocampal nerve
terminals of aged but not young rats. Finally, we observed a significant
increase of both the binding density of [3H]CGS 21680 and the
[3H]ZM241385 as well as of the anti-A2A receptor
immunoreactivity in hippocampal nerve terminal membranes from aged compared
with young rats. This shows that A2A receptor-mediated facilitation
of hippocampal synaptic transmission is larger in aged than young rats due to
increased A2A receptor density in nerve terminals and to the
modified transducing system operated by A2A receptors, from a
protein kinase C mediated control of A1 receptors into a direct
protein kinase A dependent facilitation of synaptic transmission. |
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INTRODUCTION |
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; Barnes et al.
2000) that results from the general age-induced compromise of
cellular function (Dani et al.
1997). The more direct approach to enhance excitatory transmission
would be to use analogues of the main excitatory neurotransmitter in the
brain, but glutamate analogues can also produce excitotoxic effects
(Rothman 1984). An indirect
approach is to interfere with neuromodulatory systems of glutamatergic
transmission since they fine-tune transmission rather than support information
processing.
One ubiquitous neuromodulatory system in the CNS is operated by adenosine.
Adenosine neuromodulation is mostly conceived as an inhibitory system that
restrains excitatory transmission through activation of inhibitory
A1 receptors (Dunwiddie and
Masino 2001). However, neurons are equipped with other adenosine
receptors, mainly A2A receptors, whose activation results in a
facilitation of neurotransmitter release
(Cunha 2001). The search of the
physiological relevance of these A2A receptors has given rise to
the idea that they mostly act to fine-tune other neuromodulatory systems
(Sebastião and Ribeiro
2000) and, among others, to control the tonic inhibitory action of
A1 receptors (Lopes et al.
1999a). Thus the control by adenosine of neurotransmitter release
should be conceived as a balance between inhibitory A1 and
facilitatory A2A receptor-mediated actions
(Cunha 2001).
This potential for adenosine to either inhibit or facilitate synaptic
transmission makes this neuromodulatory system a likely candidate to be reset
to compensate the age-related changes in neuronal performance. In fact,
previous studies have shown a decrease in the density of A1
receptors and an increase in the density of A2A receptors in the
limbic cortex and neocortex of aged rats
(Cunha et al. 1995a). This
decrease in the density of A1 receptors is accompanied by a
decreased ability of A1 receptor agonists to inhibit synaptic
transmission in the hippocampus of aged rats
(Sebastião et al.
2000). Also, the increased density of A2A receptors is
accompanied by a greater efficiency of A2A receptors to enhance
acetylcholine release from hippocampal preparations
(Lopes et al. 1999b). However,
the role of A2A receptors in the control of acetylcholine release
appears to be different from the A2A receptor-mediated facilitation
of glutamatergic transmission in young adult rats. Thus the A2A
receptor-mediated facilitation of acetylcholine release is independent of
A1 receptor function (Cunha et
al. 1995b) and involves activation of protein kinase A
(Rebola et al. 2002), whereas
the A2A receptor-mediated facilitation of glutamatergic
transmission results from a desensitization of tonic A1
receptor-mediated inhibition of synaptic transmission
(Lopes et al. 2002) and
involves activation of protein kinase C
(Cunha and Ribeiro 2000). Thus
we now investigated if the A2A receptor-mediated modulation of
synaptic transmission is modified in hippocampal slices of aged rats.
We found that there is an increased ability of A2A receptors to
facilitate synaptic transmission in the hippocampus of aged rats and that this
effect of A2A receptors in aged rats is no longer due to a protein
kinase C mediated attenuation of A1 receptor tonic inhibition
(Cunha and Ribeiro 2000; Lopes
et al. 1999a,
2002) but rather to a protein
kinase A mediated direct facilitation of synaptic transmission.
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METHODS |
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Adenosine, 2-chloroadenosine, CGS21680, chelerythrine, H-89, and ZM241385 were made up to a 5 mM stock solution in dimethylsulfoxide; rolipram was made up in a 50 mM stock solution in dimethylsulfoxide and DPCPX was made up into 5 mM stock in 99% dimethylsulfoxide and 1% NaOH 1 M. These stock solutions were aliquoted and stored at –20°C and aqueous dilution of these stock solutions was made daily.
Animals
Male Wistar rats from the Gulbenkian Institute animal house or from Harlan
Iberica were used throughout this study. Young adult rats were 6 weeks old
(140–160 g), whereas aged rats were 21–24 mo old (950–1,080
g). In some experiments, 18-mo-old rats were used (960–1,020 g). Most
studies in purinergic modulation have been performed in 6-weeks-aged rats,
which are considered juvenile or young adult rats, whereas 18- to 24-mo-old
rats are considered aged rats (i.e., close to the limit of life expectancy;
Masoro 1991;
Miller and Nadon 2000).
Indeed, around 30% of the initial population of rats allocated for this study
did not reach this age range, confirming by operational criteria that rats
18–24 mo old are aged rats (see
Masoro 1991). In neither group
of aged rats was there any evidence of gross anatomical lesions in the brain.
The handling and use of these animals was according the EU guidelines for use
of experimental animals, the animals being anesthetized under halothane
atmosphere before being killed by decapitation. The number of animals used was
kept to the absolute minimum, with care not to use more animals than these
required to obtain statistically significant results.
Electrophysiological recordings of hippocampal synaptic transmission
One 400-µm hippocampal slice, obtained as previously described (e.g.,
Lopes et al. 1999a), was
transferred to a 1-ml recording chamber for submerged slices and continuously
superfused, at a flow rate of 3 ml/min, with gassed (95% O2-5%
CO2) Krebs solution, kept at 30°C, of the following composition
(in mM): 125 NaCl, 3 KCl, 1.25 NaH2PO4, 25
NaHCO3, 2 CaCl2, 1 MgSO4, and glucose 10, pH
7.4. Drugs were added to this superfusion solution. Electrophysiological
recordings of field excitatory postsynaptic potentials (fEPSP) were obtained
as previously described (e.g.,
Sebastião et al. 2000).
Stimulation (rectangular pulses of 0.1 ms applied once every 15 s) was
delivered through a bipolar concentric electrode placed on the Schaffer
fibers, in the stratum radiatum near the CA3/CA1 border. Orthodromically
evoked fEPSPs were recorded through an extracellular microelectrode (4 M NaCl,
2–5 M
resistance) placed in the stratum radiatum of the CA1 area.
The intensity of the stimulus [which was similar in hippocampal slices from
young adult (220 ± 40 µA, n = 7) and aged rats (290
± 30 µA, n = 14)] was adjusted to evoke a fEPSP with an
amplitude of 0.7–1 mV without appreciable population spike
contamination. Recordings were obtained with an Axoclamp 2B amplifier coupled
to a DigiData 1200 interface (Axon Instruments, Foster City, CA) and averages
of eight consecutive responses were continuously monitored on a personal
computer with the LTP 1.01 software
(Anderson and Collingridge
1997). Responses were quantified as the initial slope of the
averaged fEPSPs and the effect of drugs, added to this superfusion solution,
was estimated by changes in the fEPSP slope compared with baseline.
A2A receptor binding to hippocampal nerve terminals membranes
[3H]CGS21680 or [3H]ZM241385 binding studies were
performed as previously described (e.g., Cunha et al.
1995a,
1997) using membranes from
hippocampal nerve terminals (see e.g.,
Lopes et al. 1999a). Briefly,
[3H]CGS21680 (60 nM) binding was for 4 h; [3H]ZM241385
(2 nM) binding was for 60 min, and [3H]SCH58261 (10 nM) was for 60
min at room temperature (23–25°C), with 282–384 µg of
membrane protein in a final volume of 300 µl in an incubation solution
containing 50 mM Tris-HCl and 10 mM MgCl2, pH 7.4, with 5 U/ml
adenosine deaminase. Specific binding was determined by subtraction of the
nonspecific binding, which was measured in the presence of 2 µM XAC. The
binding reactions were stopped by vacuum filtration through Whatman GF/C glass
fiber filters, followed by washing of the filters and reaction tubes with 10
ml of the incubation solution, kept at 4°C. The filters were then placed
in scintillation vials, and 5 ml of scintillation liquid (Scintran Cocktail T,
Wallac, Pharmacia-Portugal) was added. Radioactivity bound to the filters was
determined after 12 h with an efficiency of 55–60% for 2 min. Binding
assays were performed in duplicate. Membrane protein was determined according
to Peterson (1977).
cAMP assays in hippocampal nerve terminals
Hippocampal synaptosomes were prepared as previously described (e.g.,
Cunha et al. 1995b) and
resuspended in 1 ml gassed Krebs solution also containing 2 U/ml adenosine
deaminase and 50 µM rolipram. A 90-µl synaptosomal aliquot was warmed at
37°C for 15 min and then incubated at 37°C for 4 min with gassed Krebs
containing adenosine deaminase and rolipram (control) or with this modified
Krebs containing isoproterenol (30 µM) or CGS21680 (30–1,000 nM)
without or with ZM241385 (20 nM). The treated synaptosomes were then
inactivated by boiling for 10 min in 1 ml of 50 mM Tris/4 mM EDTA, pH 7.6. The
levels of cAMP in the supernatants obtained after sonication and
centrifugation (14,000 g, 10 min, 4°C) were quantified with a
radioimmunoassay kit (Amersham), as previously described
(Lopes et al. 1999b), and
protein was measured according to Peterson
(1977).
Western blot analysis of A2A receptor immunoreactivity in hippocampal nerve terminals
The analysis of adenosine A2A receptor immunoreactivity was
carried out as previously described
(Rebola et al. 2002) in
membranes from a Percoll-purified synaptosomal fraction of the hippocampus
(e.g., Lopes et al. 1999a).
Briefly, after determining the amount of protein
(Spector 1978), each sample
was diluted with two volumes of a solution containing 8 M urea, 100 mM
dithiothreitol, 2% (wt/vol) sodium dodecyl sulfate, and 375 mM Tris-HCl pH 6.8
and incubated for 2 h at 37°C. These diluted samples and the prestained
molecular weight markers (Amersham) were separated by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (10% with a 4% concentrating
gel) under reducing conditions and electro-transferred to polyvinylidene
difluoride membranes (0.45 µm from Amersham). After blocking for 2 h at
room temperature with 5% milk in Tris-buffered saline, pH 7.6 containing 0.1%
Tween 20 (TBS-T), the membranes were incubated overnight at 4°C with the
goat IgG anti-adenosine A2A receptor antibody (1:100 dilution).
After four washing periods for 10 min with TBS-T containing 0.5% milk, the
membranes were incubated with the alkaline phosphatase conjugated rabbit
anti-goat IgG secondary antibody (1:2,000 dilution from Calbiochem) in TBS-T
containing 1% milk for 90 min at room temperature. After five 10-min washes in
TBS-T containing 0.5% milk, the membranes were incubated with Enhanced
Chemi-Fluorescence for 5 min and then analyzed with a Storm (Molecular
Devices).
Statistics
The values presented are mean ± SE of n experiments. To
test the significance of the effect of a drug versus control, a paired
Student's t-test was used. When making comparisons from different
sets of experiments with control, ANOVA was used, followed by Dunnett's test.
P 
0.05 was considered to represent a significant difference.
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RESULTS |
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Since there is an increase in the number of CGS21680 binding sites in the
hippocampus of aged rats (Cunha et al.
1995a), we investigated whether there was also an increased
efficiency of A2A receptors to modulate hippocampal synaptic
transmission in aged rats. A single concentration (10 nM) of the
A2A receptor agonist, CGS21680, was tested, since it produces a
maximal facilitation of synaptic transmission in young adult animals
(Cunha et al. 1997). In the
present set of experiments, CGS21680 (10 nM) caused a facilitation of 19
± 2% (n = 5) in young adult rats and a greater facilitation
(38 ± 6%, n = 8, P < 0.05) of fEPSP slope in aged
rats (Fig. 1, A and
B). To compare the effect of CGS21680 between the two age
groups, a similar intensity of stimulation was used, which caused a smaller
fEPSP slope and amplitude in aged rats, as previously observed (e.g.,
Bauman et al. 1992;
Sebastião et al. 2000);
and, although input/output curves were not performed in the two age groups,
previous studies in young adult rats suggested that the CGS21680-induced
facilitation of synaptic transmission is apparently not related to the
strength of stimulation (see Cunha et al.
1997; Cunha and Ribeiro
2000; Lopes et al.
1999a,
2002). In 18-mo-old rats,
CGS21680 (10 nM) caused a 41 ± 4% (n = 4) facilitation of
fEPSP slope, and, as illustrated in Fig.
1C, this effect of CGS21680 was prevented by the
adenosine A2A receptor antagonist, ZM241385 (20 nM, n =
3), as has been shown to occur in young adult animals
(Cunha et al. 1997). ZM241385
(20 nM) was devoid of effects on synaptic transmission in young rats, as well
as in 18-mo-old rats (n = 3). It should be pointed out that although
ZM241385 is the best A2A receptor antagonist commercially
available, it is not an ideal tool to probe the role of tonic A2A
receptor activation by endogenous adenosine since it also interferes with
A1 receptor-mediated responses
(Lopes et al. 1999c), although
it only interferes with A1 receptor binding in micromolar
concentrations (Cunha et al.
1997).
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Although in young adult rats it is possible to record facilitatory
A2A receptor-mediated effects on synaptic transmission, these
facilitatory effects are not due to a direct facilitation of synaptic
transmission, but rather to an A2A receptor-induced decrease of
tonic A1 receptor-mediated inhibition of synaptic transmission
(Lopes et al. 1999a,
2002). Accordingly,
superfusion of hippocampal slices from young adult rats with adenosine causes
a concentration-dependent inhibition of synaptic transmission that is blocked
by effective concentrations of A1 receptor antagonists, such as
DPCPX (e.g., Dunwiddie and Diao
1994). As occurs in young adult rats, superfusion of hippocampal
slices of aged rats with adenosine (50 µM) also produced a nearly complete
blockade of synaptic transmission (Fig.
2). However, in contrast with what occurs in young adult rats
(Fig. 2A), in the
presence of DPCPX (50 nM), adenosine now increased the fEPSP slope in
hippocampal slices of aged rats (57.4 ± 3.9%, n = 4;
P < 0.05) (Fig.
2B). A close chemical analogue of adenosine that is an
agonist of all adenosine receptors and is less prone to metabolism,
2-chloroadenosine (10 µM), also facilitated (66.9 ± 7.4%, n
= 3, P < 0.05) fEPSP slope in the presence of DPCPX (50 nM) in
aged rats, excluding the possibility that modifications of adenosine
metabolism in aged rats (Cunha et al.
2001) might be responsible for this effect
(Fig. 2B). These
facilitatory effects of adenosine and 2-chloroadenosine recorded in the
presence of DPCPX (50 nM) in aged rats were attenuated by ZM241385 (20 nM),
indicating the involvement of A2A receptor activation
(Fig. 2, B and
C).
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This observation that adenosine is able to facilitate synaptic transmission
in aged rats on blockade of inhibitory A1 receptors further
supports a greater functional impact of A2A receptor activation in
aged rats. Also, it indicates that the mechanism by which A2A
receptors facilitate synaptic transmission is different in aged and in young
adult rats. In fact, in young adult rats, the A2A receptor-induced
facilitation of synaptic transmission is blocked on removal of tonic
A1 receptor-mediated inhibition of synaptic transmission (Lopes et
al. 1999a,
2002), whereas in aged rats
the A2A receptor-mediated facilitation of synaptic transmission is
independent of A1 receptor function. Since A2A receptors
can couple to different G proteins in different systems
(Cunha 2001), we explored the
possibility that there was a difference in the transducing systems operated by
A2A receptors in young adult and in aged rats.
Effect of A2A receptor activation on cAMP levels in hippocampal nerve terminals
In young adult rats, A2A receptor-mediated facilitation of
hippocampal synaptic transmission is protein kinase C dependent, but not
protein kinase A dependent (Cunha and
Ribeiro 2000). However, in several other systems, A2A
receptors facilitate neurotransmitter release on increasing cAMP levels in
nerve terminals (Correia-de-Sá and
Ribeiro 1994; Gubitz et al.
1996; Okada et al.
2001). Activation of A2A receptors causes a greater
accumulation of cAMP in cortical slices of aged compared with young adult rats
(Lopes et al. 1999b), but it
is not known if this also occurs in hippocampal nerve terminals.
Since we had previously observed that the concentration range required to
detect CGS21680-induced cAMP increases is nearly 10-fold greater than that
required to facilitate neurotransmitter release in other CNS preparations
(Lopes et al. 1999b), we
tested the effect of higher concentrations (30–1,000 nM) of CGS21680 on
cAMP levels. As shown in Fig.
3A, CGS21680 (30–1,000 nM) failed to modify cAMP
levels in rat hippocampal nerve terminals of young adult rats. In contrast, in
aged rats, CGS21680 enhanced cAMP levels in hippocampal nerve terminals from a
basal level of 5.2 ± 0.9 pmol/mg protein (n = 4) to a maximal
level of 12.2 ± 1.1 pmol/mg protein (n = 4) at 300 nM CGS21680
(Fig. 3A). This
stimulatory effect of CGS21680 was mediated by A2A receptor
activation since ZM241385 (20 nM) attenuated the stimulatory effect of
CGS21680 (300 nM) on cAMP levels in hippocampal nerve terminals of aged rats
(Fig. 3A). It should
be noted that the basal levels of cAMP were lower in nerve terminals of aged
compared with young adult rats, as previously observed in different brain
preparations (Lopes et al.
1999b; Zimmerman and Berg
1974). This is an indication that the tonic activation of
A2A receptors is not a receptor system responsible for the control
of basal cAMP levels in hippocampal nerve terminals.
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To test if this A2A receptor-induced increase in cAMP was due to
an increased efficiency of A2A receptors rather than to a general
responsiveness of adenylate cyclase-coupled receptor in hippocampal nerve
terminals from aged rats, we compared the ability of the
–adrenergic receptor agonist, isoproterenol, to increase cAMP
levels in hippocampal nerve terminals of young adult and aged rats, since
–receptor activation increases cAMP in rat cortical nerve
terminals (Herrero and Sanchez-Prieto
1996). As shown in Fig.
3B, isoproterenol (30 µM) caused a nearly similar
increase of cAMP levels in young adult (58.2 ± 5.2 pmol/mg protein,
n = 4) and in aged rat hippocampal nerve terminals (46.6 ± 4.1
pmol/mg protein, n = 4).
Transducing system operated by A2A receptors to facilitate synaptic transmission in aged rats
The findings that A2A receptor-mediated facilitation of
hippocampal synaptic transmission in aged rats occurred independently of
A1 receptors and that A2A receptors triggered cAMP
accumulation in aged hippocampal nerve terminals led us to test if
A2A receptors facilitated hippocampal synaptic transmission in aged
rats via an adenylate cyclase/cAMP/protein kinase A pathway, or through a
protein kinase C pathway, as has been observed in young adult rats
(Cunha and Ribeiro 2000).
Figure 4A shows time-course recordings of synaptic transmission in hippocampal slices from aged rats that illustrate the ability of the protein kinase A inhibitor, H-89 (1 µM), to prevent the facilitatory effects of CGS21680 (10 nM), and the ability of the protein kinase C inhibitor, chelerythrine (6 µM), only to attenuate the facilitatory effect of CGS21680 (10 nM). In the average of three experiments, CGS21680 (10 nM) caused a 67.6 ± 18.7% facilitation of fEPSP slope and this effect was attenuated by 26.5 ± 9.5% by chelerythrine (6 µM) and by 81.2 ± 2.1% by H-89 (1 µM) (Fig. 4B). In a fourth experiment in aged rats, CGS21680 increased fEPSP slope by 48% and this facilitatory effect was attenuated by 91% by H-89 (1 µM) and only by 16% by another protein kinase C inhibitor, bisindolylmaleimide (1 µM). By themselves, chelerythrine (6 µM) inhibited by 16 ± 3% fEPSP slope in aged rats, whereas H-89 (1 µM) was virtually devoid of effects (5 ± 4% inhibition of fEPSP slope, n = 3).
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Increase in the density of A2A receptors in hippocampal nerve terminals of aged rats
The initial reason that led us to test if there was an increase
facilitatory effect of A2A receptor activation on hippocampal
synaptic transmission was the observation that there was an increased density
of A2A receptors in the hippocampus of aged rats
(Cunha et al. 1995a). This
increase was measured in whole membranes, whereas the A2A receptor
facilitation of fEPSPs and of cAMP levels in aged rats results from activation
of presynaptic A2A receptors. Thus it remained to be established if
there was also a proportionally greater increase in the density of
A2A receptors in hippocampal nerve terminals of aged rats. For that
purpose, we initially carried out binding studies with an agonist and
antagonist of A2A receptors, [3H]CGS21680 (60 nM) and
[3H]ZM241385 (2 nM) (Cunha et
al. 1997), in membranes derived from synaptosomes prepared from
the hippocampus of young adult and aged rats. The data summarized in
Fig. 5, A and
B, show that there was an increased density of both
[3H]CGS21680 (60 nM) and [3H]ZM241385 (2 nM) binding to
hippocampal nerve terminal membranes of aged compared with young adult rats
(n = 4), which is proportionally greater than that occurring in whole
hippocampal membranes (Cunha et al.
1995a).
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Finally, we confirmed this increased density of A2A receptors in membranes of hippocampal nerve terminals of aged rats using Western blot analysis. As illustrated in Fig. 5C, there was a more intense immuno-reactivity of the anti-adenosine A2A receptor antibody in hippocampal nerve terminals from aged compared with young adult rats. In three Western blots carried out with membranes from three different groups of young adult and aged rats, we observed a 76 ± 6% increase in the densitometrically measured immuno-reactivity in aged compared with young adult rats.
Age-dependent increase in A2A receptor binding and A2A receptor-induced cAMP accumulation
The results presented so far indicate that there is an increased density of A2A receptors and a change in the transducing system operated by A2A receptors in the hippocampus of aged rats. However, given that 20 mo is a relatively long time window in a rat, it is not clear if the changes observed for the neuromodulatory system operated by A2A receptors result from a gradual change over life span or to a switch occurring essentially in the aged rats. To address this question, we investigated the changes in the density of A2A receptors and in the ability of A2A receptor activation to increase cAMP levels in nerve terminals from the hippocampus of rats at 0.5, 2, 6, 12, 18, and 24 mo.
As illustrated in Fig. 6A, the binding A2A receptor antagonist, [3H]SCH58261 (10 nM), was not statistically different (P > 0.05) in hippocampal nerve terminals membranes derived from animals with ages ranging from 0.5 to 12 mo. However, the specific binding of [3H]SCH58261 (10 nM) was significantly (P < 0.05) larger in hippocampal nerve terminals membranes from 18-mo-old rats and did not further increase in rats at 24 mo (Fig. 6A). Likewise, there was no significant (P > 0.05) change induced by CGS21680 (300 nM) in the levels of cAMP in nerve terminals from 0.5- to 6-mo-old rats. In 12-mo-old rats, CGS21680 (300 nM) caused a small but significant (P < 0.05) increase in the levels of cAMP (Fig. 6B). However, this CGS21680 (300 nM)-induced increase in the levels of cAMP was considerably greater in hippocampal nerve terminals derived from 18- and 24-mo-old rats (Fig. 6B).
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DISCUSSION |
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;
Lopes et al. 2002), all the
experiments were carried out by the same experimenter and in the same set-up
with a 3- to 7-mo time span, thus limiting possible bias of such
cross-experiment comparisons. It is of particular interest to note that,
although most of the study was designed to investigate the status of the
neuromodulatory system operated by A2A receptors in the hippocampus
of aged rats in comparison to young adult rats, the study of the changes on
aging of A2A receptor density and of its efficiency to trigger cAMP
accumulation in hippocampal nerve terminals suggests that these changes might
essentially be a feature of the aging process rather than a slow continuous
process that evolves through maturation of the animals.
Adenosine A2A receptors were initially described as being
coupled to the GS/adenylate cyclase/cAMP/protein kinase A pathway
(Fredholm et al. 1994).
However, several studies have shown that A2A receptors can couple
to different transducing systems in different preparations, making it clear
that A2A receptors are potentially pleiotropic receptors (reviewed
in Cunha 2001). Two
possibilities can be considered to understand the age-related change in the
transducing system operated by limbic cortical A2A receptors. One
would rely on a change in the density of GS proteins in aged
hippocampal nerve terminals, whereas the second would rely on an increase in
the number of A2A receptors. There is a slight decrease in the
density of GS proteins in the cortex of aged individuals
(Young et al. 1991), but there
is a general trend for a preservation of the efficiency to trigger cAMP
formation in the cortex or hippocampus of aged animals
(Sugawa and May 1993;
Zimmerman and Berg 1975).
Also, the first hypothesis is not supported by the observation that the
–receptor agonist, isoproterenol, caused a similar increase in
cAMP levels in hippocampal nerve terminals of aged compared with young adult
rats, in accordance with the preserved function of –receptors in
the cortex or hippocampus of aged animals
(Dierssen et al. 1992). In
contrast, the present observation that there was a nearly doubling of the
binding density of A2A receptors in hippocampal nerve terminals of
aged compared with young adult rats makes it possible that the change in
receptor number could be responsible for the change in the main transducing
system operated by A2A receptors in aged animals. In fact,
manipulation of the levels of different receptors in heterologous expression
systems has shown that different transducing systems can be recruited
according to the density and level of activation of these receptors (reviewed
in Gudermann et al. 1997).
Although little is known on the trafficking and subcellular location of
A2A receptors (reviewed in
Fredholm et al. 2002), it is
possible that the small number of A2A receptors in hippocampal
nerve terminals of young adult rats may be scaffolded with pathways directed
for a protein kinase C mediated attenuation of A1 receptor function
(Cunha and Ribeiro 2000; Lopes
et al. 1999a,
2002), whereas the increase in
the number of A2A receptors in hippocampal nerve terminals of aged
rats would allow their more generally recognized coupling to the adenylate
cyclase/cAMP/protein kinase A pathway. Furthermore, one needs to consider the
possibility that covalent modifications of the A2A receptor, which
has the longest C-terminal tail among adenosine receptors
(Svenningsson et al. 1999),
may control the G protein coupling of A2A receptors, an issue that
has not yet been experimentally tested.
Of direct relevance for the functional importance of the changes in the
adenosine neuromodulatory system in aged rats is the conclusion that
A2A receptors appear to be a direct facilitatory system in aged
rats compared with their predominant fine-tuning role in young adult rats
(Sebastião and Ribeiro
2000). Thus whereas in young adult rats the role of hippocampal
A2A receptors is mainly to control A1 receptor function,
in the aged rats, activation of A2A receptors causes a direct
facilitation of glutamatergic transmission. Interestingly, this occurs in
parallel with a decreased density of A1 receptors
(Cunha et al. 1995a),
resulting in a decreased potency of A1 receptor agonists to
modulate hippocampal synaptic transmission
(Sebastião et al.
2000). Thus the adenosine neuromodulatory system, which results
from a balanced activation of inhibitory A1 and facilitatory
A2A receptors (Cunha
2001), appears to be unbalanced toward facilitatory A2A
receptors in aged animals so as to compensate the lower neuronal efficiency in
the CA1 area of the rat hippocampus (Barnes
1994; Barnes et al.
2000). This raises the suggestion that the adenosine
neuromodulatory system may be an adaptation system to compensate for the
changes in synaptic transmission that occur on aging.
A modified A2A receptor function has also been proposed to occur
in the striatum (Corsi et al.
1999,
2000;
Popoli et al. 1998a). It is
important to keep in mind that the main subcellular localization and
functional role of striatal A2A receptors are different from
extra-striatal A2A receptors
(Fredholm et al. 2002). In
fact, whereas in extra-striatal areas the role of neuronal A2A
receptors is mostly a facilitation of neurotransmitter release (reviewed in
Cunha 2001), striatal
A2A receptors have a main postsynaptic location controlling the
function of D2 receptors
(Svenningsson et al. 1999) and
also of NMDA receptors (Nash and Brotchie
2000). Overall, the evolution on aging of striatal and hippocampal
or cortical A2A receptors appears to be in opposite directions.
Indeed, binding studies revealed a decreased binding density of A2A
receptors in the striatum of aged rats
(Cunha et al. 1995a;
Popoli et al. 1998b) that is
accompanied by a decreased expression of A2A receptor mRNA
(Schiffmann and Vanderhaeghen
1993), precisely the opposite of what occurs for A2A
receptors in the hippocampus and cortex of aged rats
(Cunha et al. 1995a;
Lopes et al. 1999b). This
opposite age-related change in the density of A2A receptors in the
striatum and in the cortex also raises some concern on the therapeutic use of
A2A receptor antagonists as anti-Parkinsonian drugs (e.g.,
Fredholm et al. 2002). In
fact, in young adult rats that are used as models for this striatal
dysfunction, blockade of central A2A receptors is roughly
equivalent to blockade of striatal A2A receptors given the high
density of striatal versus extra-striatal A2A receptors
(Svenningsson et al. 1999).
However, Parkinson's disease is prevalent in aged individuals and we presently
observed that there is a marked increase in the density and functional impact
of A2A receptors in the hippocampus and in the cortex (see also
Lopes et al. 1999b), whereas
the opposite is observed in the striatum
(Cunha et al. 1995a;
Popoli et al. 1998b). This
brings to stage the possible more evident side effects of long-term
administration of A2A receptor antagonists in the elderly, namely
in view of the observed effects of A2A receptors in the control of
hippocampal synaptic plasticity (de
Mendonça and Ribeiro 1994), sleep
(Satoh et al. 1999), and
psychosis (Ferré et al.
1994; Sills et al.
2001).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests: Rodrigo A. Cunha, Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal (E-mail: racunha{at}clix.pt).
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INTRODUCTION
