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Keywords:

  • adenosine;
  • caffeine;
  • dopamine;
  • glutamate;
  • microdialysis;
  • NMDA receptor;
  • nucleus accumbens

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Adenosine, by acting on adenosine A1 and A2A receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A1 inhibiting and activation of A2A receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A1 and A2A receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 µm), the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 µm), the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 µm), or the non-selective A1-A2A receptor antagonist in vitro caffeine (300, 1000 µm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 µm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A2A receptor antagonist MSX-3 (1 µm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A1 and A2A receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A1 vs. A2A receptor antagonism in the central effects of caffeine.

Abbreviations used
APV

dl-2-amino-5-phosphonovaleric acid

CPT

8-cyclopentyl-1,3-dimethylxanthine

NAc

nucleus accumbens

NMDA

N-methyl-d-aspartate

VTA

ventral tegmental area

The ventral part of the striatum, which is made of the ventromedial caudate-putamen, the nucleus accumbens (NAc), with its two compartments shell and core, and the olfactory tubercle, is involved in the elicitation of behavioral responses to motivational stimuli, that is, the conversion of motivation into action (Mogenson et al. 1980). Dopaminergic and glutamatergic afferents are the two main input systems to the striatum. The NAc receives its dopaminergic input from the ventral tegmental area (VTA) and its glutamatergic input from the basolateral nucleus of the amygdala, the ventral hippocampus, the medial prefrontal cortex and the midline thalamic nuclei, especially the paraventricular nucleus (Groenewegen et al. 1999). These dopaminergic and glutamatergic inputs converge in the dendritic spines of GABAergic striatal efferent neurons (Totterdell and Smith 1989; Sesack and Pickel 1990, 1992; Johnson et al. 1994; Pinto et al. 2003), which constitute more than 90% of the striatum's neuronal population (Smith and Bolam 1990). Glutamatergic synapses are located on the head of dendritic spines, while the dopaminergic afferents usually are located on the neck of dendritic spines. This allows dopamine to play a major modulatory role in the excitatory effects of glutamate in dendritic spines (Smith and Bolam 1990; Centonze et al. 2001).

In addition to this postsynaptic dopamine-mediated modulation of glutamatergic neurotransmission, there is substantial evidence for the existence of functionally significant presynaptic glutamate-mediated modulation of striatal dopaminergic neurotransmission, with stimulation of presynaptic ionotropic glutamate hetero-receptors inducing dopamine release (Roberts and Anderson 1979; Desce et al. 1992; Jin and Fredholm 1997; Segovia et al. 1997; Taepavarapruk et al. 2000; Kulagina et al. 2001; Segovia and Mora 2001; Howland et al. 2002). In the NAc this glutamate–dopamine interaction seems to depend primarily on N-methyl-d-aspartate (NMDA) receptors (Segovia and Mora 2001). As no synaptic connections seem to exist between striatal glutamatergic and dopaminergic cell terminals, this glutamate–dopamine interaction probably depends on volume transmission by spillover of glutamate from glutamatergic synapses and stimulation of extrasynaptic NMDA receptors localized in dopaminergic cell terminals (Gracy and Pickel 1996; Tarazi et al. 1998).

In addition to dopamine and glutamate, the neuromodulator adenosine plays an important role in the function of striatal GABAergic efferent neurons (Ferréet al. 1997). Striatal extracellular adenosine originates mostly from release of intracellular adenosine and from release and extracellular breakdown of cAMP (by ecto-5′-nucleotidase and phosphodiesterases) (Manzoni et al. 1998; Latini and Pedata 2001). Thus, adenosine can be produced by metabolically active cells (high ATP consumption) or cells that accumulate cAMP (stimulation of receptors positively linked to adenyyl-cyclase). Of the four known adenosine receptors (A1, A2A, A2B and A3), adenosine A1 and A2A receptors are primarily responsible for the central effects of adenosine (Fredholm et al. 2001a). Adenosine A2A receptors are highly concentrated in the striatum, where ultrastructural analysis has shown that they are concentrated in the dendritic spines of one subtype of GABAergic striatal efferent neuron, the striatopallidal neuron, especially in the vicinity of glutamatergic synapses (Hettinger et al. 2001; Rosin et al. 2003). Adenosine A2A receptors are also found presynaptically in glutamatergic but not in dopaminergic terminals, although with lower density (Hettinger et al. 2001; Rosin et al. 2003). Adenosine A1 receptors are more widespread than A2A receptors and they are found in all striatal neuronal elements, mostly in glutamatergic terminals (Wojcik and Neff 1983; Alexander and Reddington 1989).

Adenosine A1 and A2A receptors exert opposite modulatory roles on extracellular levels of glutamate and dopamine in the striatum, with activation of A1 receptors inhibiting and stimulation of A2A receptors stimulating glutamate and dopamine release (Wood et al. 1989; Lupica et al. 1990; Zetterstrom and Fillenz 1990; Ballarin et al. 1995; Popoli et al. 1995; Okada et al. 1996; Golembiowska and Zylewska 1997; Corsi et al. 2000; Solinas et al. 2002; Karcz-Kubicha et al. 2003a; Quarta et al. 2004). As they are both found in striatal glutamatergic terminals, A1 and A2A receptors are in a position to directly modulate glutamate release. The role presynaptic A1 receptors play in mediating the inhibition of glutamatergic neurotransmission induced by adenosine is well established (Goodman et al. 1983; Yawo and Chuhma 1993; Ambrosio et al. 1997; Flagmeyer et al. 1997; Wu and Saggau 1997; Lopes et al. 2002; Kimura et al. 2003; Moore et al. 2003). Similarly, presynaptic A2A receptors localized at glutamatergic terminals are probably involved in the facilitation and inhibition of striatal glutamate release induced by A2A receptor agonists and antagonists, respectively (Popoli et al. 1995, 2003; Corsi et al. 2000; Quarta et al. 2004).

The mechanisms underlying A1 and A2A receptor-mediated modulation of striatal dopamine release are less clear than those involved in glutamate release. Although it is generally accepted that A1 receptors are presynaptically localized in dopaminergic cell terminals, the available morphological evidence is still indirect. Thus, this evidence is based on the demonstration of A1 receptor protein or mRNA in the substantia nigra and ventral tegmental area (Mahan et al. 1991; Rivkees et al. 1995; Glass et al. 1996). Furthermore, the lesioning of glutamatergic, but not dopaminergic, striatal afferents significantly decreases striatal A1 receptor function and agonist binding (Wojcik and Neff 1983; Alexander and Reddington 1989). Therefore, if A1 receptors are localized in dopaminergic cell terminals, they are in very low numbers. As, in addition, there are no A2A receptors in dopaminergic cell terminals (Hettinger et al. 2001; Rosin et al. 2003), the main mechanism underlying adenosine-mediated modulation of striatal dopamine release should be mostly indirect. The results of the present in vivo microdialysis study indicate that modulation of dopamine release in the NAc by both A1 and A2A receptors is mostly secondary, due to their opposite modulatory role on glutamatergic neurotransmission and depends on the stimulation NMDA receptors.

Subjects and drugs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Male Sprague–Dawley rats, weighing 300–350 g, were used in all experiments. Animals, housed in groups of two, were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care; all experimentation was conducted in accordance with the guidelines of the Institutional Care and Use Committee of the Intramural Research Program, National Institute on Drug Abuse (NIDA), National Institutes of Health, the directives of the Principles of Laboratory Animal Care (National Institutes of Health publication number 85–23, revised 1985). Caffeine (caffeine anhydrate base), the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) and the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT) were purchased from Sigma (St. Louis, MO, USA), NMDA and the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV) were purchased from Tocris (Ellisville, MO, USA) and the A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3) was synthesized by the Pharmaceutical Institute, University of Bonn, Germany.

In vivo microdialysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Concentric microdialysis probes were prepared as described previously (Pontieri et al. 1996). Animals were anesthetized with Equithesin (NIDA Pharmacy, Baltimore, MD, USA), and probes were implanted in the shell of the NAc (coordinates with respect to bregma: anterior, + 2.2; lateral, − 1.0; ventral, − 7.5 from the dura). The experiments were performed on freely moving rats 24 h after probe implantation. A Ringer solution (in mm) of 147 NaCl, 4 KCl, and 2.2 CaCl2 was pumped through the dialysis probe at a constant rate of 1 µL/min. All drugs were freshly dissolved in the Ringer solution and pH was corrected when necessary. After a washout period of 90 min, samples were collected at 20-min intervals and split into two fractions of 10 µL, to separately measure glutamate and dopamine content. Each animal was used to study the effect of one treatment by local administration (perfusion by reverse dialysis). At the end of the experiment, rats were killed with an overdose of Equithesin and methylene blue was perfused through the probe. The brain was removed and placed in a 10% formaldehyde solution, and coronal sections were cut to verify probe location. Dopamine content was measured by reverse high-performance liquid chromatography (HPLC) coupled to an electrochemical detector, as described in detail previously (Pontieri et al. 1996). Glutamate content was measured by HPLC coupled to a fluorimetric detector as described in detail elsewhere (Quarta et al. 2004). Dopamine and glutamate values were transformed as percentage of basal values (mean of the three values before the drug perfusion) and transformed values were statistically analyzed with Friedman test followed by Dunn's multiple comparisons test, using GraphPad Prism 4.0 software (San Diego, CA, USA).

NMDA receptor-mediated dopamine release in the shell of the NAc

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Basal extracellular levels of dopamine and glutamate in the shell of the NAc were 4.1 ± 0.2 nm (n = 87) and 3.5 ± 0.4 µm (n = 65). In order to determine the possible involvement of NMDA receptors in the modulation of dopamine release by A2A receptor stimulation or A1 receptor blockade in the NAc, we first searched for an appropriate concentration of the competitive NMDA receptor antagonist APV. The perfusion of a relatively low concentration of NMDA (10 µm but not 3 µm) produced a significant increase in the extracellular concentration of dopamine (more than 50% with respect to basal levels) and did not modify glutamate extracellular levels in the shell of the NAc (Fig. 1). The NMDA antagonist APV (100 µm), but not the A2A receptor antagonist MSX-3 (1 µm), counteracted the effect of NMDA and did not have any significant effect when perfused alone (Fig. 1). The extracellular concentration of glutamate could not be measured during perfusion with APV, because of the disruption of the chromatographic analysis.

image

Figure 1. Extracellular concentrations of dopamine (DA) and glutamate (Glu) in the shell of the NAc after local administration of NMDA (3 or 10 µm), the NMDA receptor antagonist APV (100 µm) and the adenosine A2A receptor antagonist MSX-3 (MSX; 1 µm) alone or in combination. The horizontal lines show the period of perfusion; the lower line corresponds to APV or MSX-3 when coperfused with NMDA. The results represent means ± SEM of the percentage of basal values of the extracellular concentrations of dopamine and glutamate (n = 5–7 per group). Basal values were the means of three values before drug perfusion. NMDA produced a significant increase in the extracellular concentration of dopamine, which was completely counteracted by APV and was not counteracted by MSX-3. NMDA did not significantly modify the extracellular concentration of glutamate; p: overall probability (Friedman test); * and **, value is significantly different (p < 0.05 and p < 0.01, respectively) compared with the basal value previous to saline or drug perfusion (Dunn's multiple comparisons test).

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Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Local perfusion of the A2A receptor agonist CGS 21680 produced a significant increase in the extracellular levels of both dopamine and glutamate in the shell of the NAc. The increase in glutamate levels was dose-dependent, with 3 and 10 µm of CGS 21680 eliciting a maximal increase of about 50 and 170% with respect to basal values, respectively (Fig. 2). The effect on dopamine was less pronounced but also significant with 10 µm of CGS 21680, with a maximal increase of about 70% with respect to basal values (Fig. 2). The effect of CGS 21680 (10 µm) on both dopamine and glutamate was completely counteracted by coperfusion with the selective A2A receptor antagonist MSX-3 at a concentration (1 µm) that did not modify glutamate or dopamine levels (Fig. 2). A higher concentration of MSX-3 (3 µm) was also found to be ineffective at modifying extracellular concentrations of dopamine and glutamate (data not shown). Furthermore, the effect of CGS 21680 on dopamine was also counteracted by coperfusion with the NMDA receptor antagonist APV (100 µm) (Fig. 2).

image

Figure 2. Extracellular concentrations of dopamine (DA) and glutamate (Glu) in the shell of the NAc after local administration of the adenosine A2A receptor agonist CGS 21680 (CGS; 3 or 10 µm), the NMDA receptor antagonist APV (100 µm) and the adenosine A2A receptor antagonist MSX-3 (MSX; 1 µm) alone or in combination. The horizontal lines show the period of perfusion; the lower line corresponds to APV or MSX-3 when coperfused with CGS 21680. The results represent means ± SEM of the percentage of basal values of the extracellular concentrations of dopamine and glutamate (n = 5–9 per group). Basal values were the means of three values before drug perfusion. CGS 21680 produced a significant increase in the extracellular concentration of dopamine, which was significantly counteracted by MSX-3 and APV. CGS 21680 also produced a significant increase in the extracellular concentration of glutamate that was completely counteracted by MSX-3; p, overall probability (Friedman test); *, ** and ***, value significantly different (p < 0.05, p < 0.01 and p < 0.001, respectively) compared to the basal value previous to saline or drug perfusion (Dunn's multiple comparisons test).

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Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

Local perfusion of the A1 receptor antagonist CPT or caffeine produced a significant increase in the extracellular levels of both dopamine and glutamate in the shell of the NAc. In both cases the increase in dopamine levels was dose-dependent, with 300 and 1000 µm of CPT or caffeine eliciting a maximal increase of about 50 and 300% with respect to basal values, respectively (Figs 3 and 4). The effect of CPT on dopamine release was less sustained than that of caffeine, which kept the same maximal increase during the whole period of perfusion. The effect on glutamate was less pronounced but also significant, with 300 and 1000 µm of CPT or caffeine eliciting a maximal increase of about 50 and 200% with respect to basal values, respectively (Fig. 4). The effect of CPT (1000 µm) or caffeine (1000 µm) on dopamine or glutamate was counteracted by the coperfusion with the selective A2A receptor antagonist MSX-3 (1 µm) or the NMDA receptor antagonist APV (100 µm) (Figs 3 and 4).

image

Figure 3. Extracellular concentrations of dopamine (DA) and glutamate (Glu) in the shell of the NAc after local administration of CPT (300 or 1000 µm), the NMDA receptor antagonist APV (100 µm) and the adenosine A2A receptor antagonist MSX-3 (MSX; 1 µm) alone or in combination. The horizontal lines show the period of perfusion; the lower line corresponds to APV or MSX-3 when coperfused with caffeine. The results represent means ± SEM of the percentage of basal values of the extracellular concentrations of dopamine and glutamate (n = 5–7 per group). Basal values were the means of three values before drug perfusion. CPT produced a significant increase in the extracellular concentration of dopamine, which was significantly counteracted by APV and MSX-3. CPT also produced a significant increase in the extracellular concentration of glutamate that was significantly counteracted by MSX-3; p: overall probability (Friedman test); * and **, value significantly different (p < 0.05, and p < 0.01, respectively) compared to the basal value previous to saline or drug perfusion (Dunn's multiple comparisons test).

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image

Figure 4. Extracellular concentrations of dopamine (DA) and glutamate (Glu) in the shell of the NAc after local administration of caffeine (300 or 1000 µm), the NMDA receptor antagonist APV (100 µm) and the adenosine A2A receptor antagonist MSX-3 (MSX; 1 µm) alone or in combination. The horizontal lines show the period of perfusion; the lower line corresponds to APV or MSX-3 when coperfused with caffeine. The results represent means ± SEM of the percentage of basal values of the extracellular concentrations of dopamine and glutamate (n = 5–7 per group). Basal values were the means of three values before drug perfusion. Caffeine produced a significant increase in the extracellular concentration of dopamine, which was significantly counteracted by APV and MSX-3. Caffeine also produced a significant increase in the extracellular concentration of glutamate that was significantly counteracted by MSX-3; p, overall probability (Friedman test); * and **, value significantly different (p < 0.05, and p < 0.01, respectively) compared with the basal value previous to saline or drug perfusion (Dunn's multiple comparisons test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References

The initial specific aim of the present study was to unravel the mechanism responsible for the previously described dopamine-releasing effects of the selective A2A receptor agonist CGS 21680 (Karcz-Kubicha et al. 2003a). In view of the lack of A2A receptors in dopaminergic cell terminals (Hettinger et al. 2001; Rosin et al. 2003), it was hypothesized that CGS 21680-induced dopamine release was dependent on the glutamate-releasing properties of striatal A2A receptors and on the ability of endogenous glutamate to increase extracellular concentrations of dopamine in the striatum (see Introduction). As this glutamate–dopamine interaction in the NAc depends primarily on the stimulation of NMDA receptors (Segovia and Mora 2001) most probably localized in dopaminergic cell terminals (Gracy and Pickel 1996; Tarazi et al. 1998), our hypothesis predicts that NMDA receptor blockade should counteract CGS 21680-induced dopamine release in the NAc. Previous studies about changes in the extracellular concentrations of dopamine and glutamate in the NAc after systemic administration of compounds active at adenosine receptors could not rule out the involvement of other brain areas. Therefore, in the present microdialysis experiments, drugs were directly perfused into the NAc by reverse microdialysis.

Our results support previous studies that indicate dopamine release in the NAc is under the stimulatory control of extracellular glutamate and depends on the activation of NMDA receptors (Segovia and Mora 2001). Thus, perfusion of NMDA (10 µm) in the shell of the NAc produced an increase in the extracellular concentration of dopamine, but not glutamate, which was completely counteracted by coperfusion of the selective competitive NMDA receptor antagonist APV at a concentration (100 µm) that did not modify dopamine levels. Although the presence of NMDA autoreceptors in striatal glutamatergic terminals has been reported (Tarazi et al. 1998; Wang and Pickel 2000), we did not observe any significant change in glutamate release with NMDA administration. Nevertheless, and important for the present study, we could discard a role for endogenous glutamate in NMDA-mediated dopamine release.

Local perfusion of the A2A receptor agonist CGS 21680 produced an increase in the extracellular concentration of dopamine in the shell of the NAc, as previously found after systemic administration (Karcz-Kubicha et al. 2003a). Furthermore, as previously found in the caudate-putamen (Popoli et al. 1995), local perfusion of CGS 21680 also produced an increase in the extracellular concentration of glutamate in the shell of the NAc. Both effects were counteracted by coperfusion of the selective A2A receptor antagonist MSX-3 at a concentration (1 µm) that did not modify glutamate or dopamine levels. As the NMDA receptor antagonist APV also counteracted CGS 21680-induced dopamine release, the results support our hypothesis that glutamate release and NMDA receptor stimulation mediate the dopamine release in the shell of the NAc induced by the A2A receptor agonist.

In addition to A2A receptor agonists, systemic or local administration of A1 receptor antagonists induces an increase in striatal extracellular concentrations of both dopamine and glutamate (Okada et al. 1996; Solinas et al. 2002; Quarta et al. 2004). Furthermore, there is evidence for the existence of an antagonistic A1-A2A receptor–receptor interaction, by which stimulation of A1 receptors decreases the effects of A2A receptor stimulation on both striatal dopamine and glutamate release (Okada et al. 1996; Karcz-Kubicha et al. 2003a; Quarta et al. 2004). The present study shows that, also in the NAc, a blockade of A1 receptors by local perfusion of the A1 receptor antagonist CPT induces glutamate and dopamine release. On the other hand, A2A receptor blockade by the local perfusion of the A2A receptor antagonist MSX-3 did not modify the extracellular concentrations of glutamate and dopamine. Our previous studies showed a small but significant decrease in extracellular levels of glutamate and dopamine in the NAc after systemic administration of MSX-3 (Quarta et al. 2004), which suggests extrastriatal A2A receptors could have been involved. Taking into account the antagonistic A1-A2A receptor–receptor interaction, the present results indicate that, in the NAc, the effects of endogenous adenosine on adenosine A1 receptors predominate, which masks the effects of adenosine on A2A receptors, as previously found in the nucleus caudate-putamen by Okada et al. (1996). Thus, in the whole striatum, endogenous adenosine, by stimulating A1 receptors, counteracts the effects produced by A2A receptor stimulation. As A2A receptors are already inhibited, blockade of A2A receptors is not expected to produce significant changes in the extracellular concentrations of glutamate and dopamine. However, the results obtained with local perfusion with CGS 21680 suggest that a sufficiently strong stimulation of A2A receptors can override the inhibition imposed by A1 receptors.

Although perfusion with the A2A receptor antagonist did not produce any effect, it counteracted the increase in the extracellular concentrations of glutamate and dopamine induced by the A1 receptor antagonist. Thus, the effects of A1 receptor blockade depend on A2A receptors. The present results suggest that the main mechanism responsible for glutamate and dopamine release induced by A1 receptor blockade is the removal of the A1 receptor-mediated inhibition of A2A receptor function, due to the existence of an antagonistic interaction between A1 and A2A receptors colocalized in glutamatergic cell terminals. This would allow endogenous adenosine to induce glutamate release through A2A receptor stimulation and dopamine release through NMDA receptor stimulation. In fact, as for CGS 21680, the coperfusion with the NMDA receptor antagonist APV counteracted dopamine release induced by CPT (Fig. 5).

image

Figure 5. Adenosine receptor-mediated mechanisms involved in the modulation of glutamate and dopamine release in the NAc. This mechanism would be the control of glutamate release by means of a functional antagonistic interaction between A1 and A2A receptors (A1R and A2AR, respectively) localized in glutamatergic cell terminals, with the extracellular levels of glutamate controlling dopamine release through stimulation of NMDA receptors (NMDAR) localized in dopaminergic cell terminals (see text). (a) Under basal conditions, the stimulation of A1 receptors would be able to counteract the weak stimulatory effect of adenosine on A2A receptors, by means of an antagonistic A2A receptor–A1 receptor interaction. (b) Under physiological conditions of stronger adenosine release, a sufficiently stronger A2A receptor stimulation could override the inhibitory effect imposed by the A1 receptor, probably involving an A2A receptor-mediated reduced affinity of A1 receptors for adenosine (see Dixon et al. 1997), and therefore induce glutamate and dopamine release. The scheme shows the striatal glutamatergic and dopaminergic cell terminals making contact with the dendritic spine of the GABAergic striatal efferent neuron.

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In the present study, local perfusion with caffeine, which is known to be a non-selective A1-A2A receptor antagonist in vitro, produced the same qualitative effects as the A1 receptor antagonist CPT. Caffeine induced glutamate and dopamine release in the shell of the NAc (as previously described after systemic administration; Solinas et al. 2002; Quarta et al. 2004) and these effects where counteracted by the coperfusion of the A2A receptor antagonist MSX-3 or the NMDA receptor antagonist APV. Therefore, these results add more experimental evidence for our recently suggested predominant A1 receptor antagonism of caffeine in vivo (Karcz-Kubicha et al. 2003b). We have also suggested that the A1 receptor mechanism takes place only after acute caffeine administration and it changes to a predominant A2A receptor antagonism with chronic caffeine administration (Karcz-Kubicha et al. 2003b). The mismatch between the in vitro and in vivo pharmacology of caffeine, with the predominant in vivo A1 receptor antagonism, still needs to be explained, but it could be related to a preferential occupancy of A1 versus A2A receptors by endogenous adenosine. In this context, adenosine has been found to be more potent at human adenosine A1 than A2A receptors transfected in a mammalian cell line (Fredholm et al. 2001b). Furthermore, physiological extracellular concentrations of inosine, a main metabolite of adenosine, seem to be able to activate A1 but not A2A receptors (Fredholm et al. 2001b).

The coexistence of both stimulatory A2A and inhibitory A1 receptors in the same nerve terminal is intriguing, particularly in view of their opposite functional effects. Nevertheless, the coexistence of A1 and A2A receptors has been previously reported in other locations, such as glutamatergic terminals in the hippocampus and cholinergic terminals in the striatal muscle and it has been suggested this is consistent with the fine-tuning neuromodulatory role of adenosine (Sebastiao and Ribeiro 2000). In view of the preferential occupancy of A1 versus A2A receptors by endogenous adenosine, it could be postulated that under basal conditions there is a predominance of A1 receptor function, which is able to inhibit the weak A2A receptor stimulation induced by endogenous adenosine. Under basal conditions, the low endogenous adenosine tone would favor an inhibition of glutamatergic neurotransmission. On the other hand, A2A receptors would play a more important role under conditions of increased adenosine release. Dixon et al. (1997) found that A2A receptor stimulation can reduce the affinity of A1 receptors for agonists in striatal synaptosomes and suggested that this could be a mechanism by which A2A receptors could override the inhibitory influence of A1 receptors. Thus, under conditions of increased adenosine release A2A receptors could override the inhibition imposed by A1 receptors, as shown in the present study by the effects of perfusion with an exogenous A2A receptor agonist (CGS 21680), and favor glutamatergic and, second, dopaminergic neurotransmission. A well-established physiological mechanism that increases the extracellular levels of adenosine is a strong glutamatergic neurotransmission. It has been shown that stimulation of glutamate receptors, particularly NMDA receptors, increases the extracellular concentrations of adenosine in the brain, including the striatum (Melani et al. 1999; Latini and Pedata 2001). We have recently described another, postsynaptic, mechanism by which A2A receptors can potentiate glutamatergic neurotransmission, which is a strong synergistic interaction between adenosine A2A and glutamate mGlu5 receptors; these form heteromeric complexes in the GABAergic striatopallidal neuron (Ferréet al. 2002). The biochemical mechanisms involved in the tight antagonistic interactions between A1 and A2A receptors colocalized in the striatal glutamatergic terminals remain to be elucidated.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and drugs
  5. In vivo microdialysis
  6. Results
  7. NMDA receptor-mediated dopamine release in the shell of the NAc
  8. Adenosine A2A receptor agonist-induced dopamine release in the shell of the NAc
  9. Adenosine A1 receptor antagonist- and caffeine-induced dopamine release in the shell of the NAc
  10. Discussion
  11. References
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