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Adenosine A₂A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation.

Shen HY, Canas PM, Garcia-Sanz P, Lan JQ, Boison D, Moratalla R, Cunha RA, Chen JF - PLoS ONE (2013)

Bottom Line: The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75.Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75.We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and glutamatergic signaling.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Lab, Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Adenosine A2A receptors (A2AR) are located postsynaptically in striatopallidal GABAergic neurons, antagonizing dopamine D2 receptor functions, and are also located presynaptically at corticostriatal terminals, facilitating glutamate release. To address the hypothesis that these two A2AR populations differently control the action of psychostimulants, we characterized A2AR modulation of cocaine-induced effects at the level of DARPP-32 phosphorylation at Thr-34 and Thr-75, c-Fos expression, and psychomotor activity using two lines of cell-type selective A2AR knockout (KO) mice with selective A2AR deletion in GABAergic neurons (striatum-A2AR-KO mice), or with A2AR deletion in both striatal GABAergic neurons and projecting cortical glutamatergic neurons (forebrain-A2AR-KO mice). We demonstrated that striatum-A2AR KO mice lacked A2ARs exclusively in striatal GABAergic terminals whereas forebrain-A2AR KO mice lacked A2ARs in both striatal GABAergic and glutamatergic terminals leading to a blunted A2AR-mediated facilitation of synaptosomal glutamate release. The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75. Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75. This distinct modulation of DARPP-32 phosphorylation was associated with opposite responses to cocaine-induced striatal c-Fos expression and psychomotor activity in striatum-A2AR KO (enhanced) and forebrain-A2AR KO mice (reduced). Thus, A2ARs in glutamatergic corticostriatal terminals and in GABAergic striatal neurons modulate the action of psychostimulants and DARPP-32 phosphorylation in opposite ways. We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and glutamatergic signaling.

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Deletion of A2AR immunoreactivity in glutamatergic terminals of forebrain-A2AR KO and GABAergic terminals of both forebrain A2AR - and striatum-A2AR KO mice.Detection and quantification of the percentage of GABAergic terminals (A, vGAT-positive) and glutamatergic terminals (B, vGluT1-positive) and from forebrain-selective-A2AR KO (fb-KO) or striatum-selective-A2AR KO (st-KO) mice and their wild type (WT) littermates (control) that are endowed with A2AR immunoreactivity. The bar graphs represent the percentage of vGluT1- or vGAT-immunopositive terminals that are also endowed with A2AR immunoreactivity (mean ± SEM, 3 fields per mouse, n = 4-6 animal per group). * p<0.05 vs corresponding WT littermates, using an unpaired Student’s t test. On the left side of each bar graph are shown representative immunocytochemistry photographs displaying the superimposed immunoreactivities of vGluT1 or vGAT (green) and of A2AR (red).
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pone-0080902-g001: Deletion of A2AR immunoreactivity in glutamatergic terminals of forebrain-A2AR KO and GABAergic terminals of both forebrain A2AR - and striatum-A2AR KO mice.Detection and quantification of the percentage of GABAergic terminals (A, vGAT-positive) and glutamatergic terminals (B, vGluT1-positive) and from forebrain-selective-A2AR KO (fb-KO) or striatum-selective-A2AR KO (st-KO) mice and their wild type (WT) littermates (control) that are endowed with A2AR immunoreactivity. The bar graphs represent the percentage of vGluT1- or vGAT-immunopositive terminals that are also endowed with A2AR immunoreactivity (mean ± SEM, 3 fields per mouse, n = 4-6 animal per group). * p<0.05 vs corresponding WT littermates, using an unpaired Student’s t test. On the left side of each bar graph are shown representative immunocytochemistry photographs displaying the superimposed immunoreactivities of vGluT1 or vGAT (green) and of A2AR (red).

Mentions: To demonstrate the selectivity of A2AR deletion in st-A2AR KO and fb-A2AR KO mice, we quantified A2AR immunoreactivity in glutamatergic (vesicular glutamate transporters type 1, vGluT1-positive) and GABAergic (vesicular GABA transporters, vGAT-positive) terminals from the striatum of st-A2AR KO, fb-A2AR KO and global A2AR knockout (gb-A2AR KO) mice as well as their corresponding wild-type (WT) littermates. Quantitative analysis revealed that A2AR immunoreactivity was depleted in GABAergic terminals from st-A2AR KO and fb-A2AR KO mice to background levels (n = 4–6 animals per group, p<0.05, unpaired Student’s t test) (Figure 1A) similar to these found in gb-A2AR KO mice (not shown). In contrast, A2AR immunoreactivity in glutamatergic terminals (about 50% of vGlut1-positive terminals contain A2AR, see [7]) was completely abolished in fb-A2AR KO mice and gb-A2AR KO mice (n = 4–6 animals per group, p<0.05, unpaired Student’s t test), but was selectively preserved in st-A2AR KO mice (n = 6, p>0.05, unpaired Student’s t test) due to the presence of presynaptic A2AR on corticostriatal terminals of extra-striatal glutamatergic neurons (Figure 1B). The preservation of presynaptic glutamatergic A2AR in st-A2AR KO mice was also consistent with the normal level of A2AR binding density in total membranes [9] and synaptosomal membranes (data not shown) of the cerebral cortex of st-A2AR KO mice. Together, these data demonstrate that A2AR in glutamatergic terminals of the striatum were selectively preserved in st-A2AR KO mice but abolished in fb-A2AR KO mice.


Adenosine A₂A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation.

Shen HY, Canas PM, Garcia-Sanz P, Lan JQ, Boison D, Moratalla R, Cunha RA, Chen JF - PLoS ONE (2013)

Deletion of A2AR immunoreactivity in glutamatergic terminals of forebrain-A2AR KO and GABAergic terminals of both forebrain A2AR - and striatum-A2AR KO mice.Detection and quantification of the percentage of GABAergic terminals (A, vGAT-positive) and glutamatergic terminals (B, vGluT1-positive) and from forebrain-selective-A2AR KO (fb-KO) or striatum-selective-A2AR KO (st-KO) mice and their wild type (WT) littermates (control) that are endowed with A2AR immunoreactivity. The bar graphs represent the percentage of vGluT1- or vGAT-immunopositive terminals that are also endowed with A2AR immunoreactivity (mean ± SEM, 3 fields per mouse, n = 4-6 animal per group). * p<0.05 vs corresponding WT littermates, using an unpaired Student’s t test. On the left side of each bar graph are shown representative immunocytochemistry photographs displaying the superimposed immunoreactivities of vGluT1 or vGAT (green) and of A2AR (red).
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Related In: Results  -  Collection

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pone-0080902-g001: Deletion of A2AR immunoreactivity in glutamatergic terminals of forebrain-A2AR KO and GABAergic terminals of both forebrain A2AR - and striatum-A2AR KO mice.Detection and quantification of the percentage of GABAergic terminals (A, vGAT-positive) and glutamatergic terminals (B, vGluT1-positive) and from forebrain-selective-A2AR KO (fb-KO) or striatum-selective-A2AR KO (st-KO) mice and their wild type (WT) littermates (control) that are endowed with A2AR immunoreactivity. The bar graphs represent the percentage of vGluT1- or vGAT-immunopositive terminals that are also endowed with A2AR immunoreactivity (mean ± SEM, 3 fields per mouse, n = 4-6 animal per group). * p<0.05 vs corresponding WT littermates, using an unpaired Student’s t test. On the left side of each bar graph are shown representative immunocytochemistry photographs displaying the superimposed immunoreactivities of vGluT1 or vGAT (green) and of A2AR (red).
Mentions: To demonstrate the selectivity of A2AR deletion in st-A2AR KO and fb-A2AR KO mice, we quantified A2AR immunoreactivity in glutamatergic (vesicular glutamate transporters type 1, vGluT1-positive) and GABAergic (vesicular GABA transporters, vGAT-positive) terminals from the striatum of st-A2AR KO, fb-A2AR KO and global A2AR knockout (gb-A2AR KO) mice as well as their corresponding wild-type (WT) littermates. Quantitative analysis revealed that A2AR immunoreactivity was depleted in GABAergic terminals from st-A2AR KO and fb-A2AR KO mice to background levels (n = 4–6 animals per group, p<0.05, unpaired Student’s t test) (Figure 1A) similar to these found in gb-A2AR KO mice (not shown). In contrast, A2AR immunoreactivity in glutamatergic terminals (about 50% of vGlut1-positive terminals contain A2AR, see [7]) was completely abolished in fb-A2AR KO mice and gb-A2AR KO mice (n = 4–6 animals per group, p<0.05, unpaired Student’s t test), but was selectively preserved in st-A2AR KO mice (n = 6, p>0.05, unpaired Student’s t test) due to the presence of presynaptic A2AR on corticostriatal terminals of extra-striatal glutamatergic neurons (Figure 1B). The preservation of presynaptic glutamatergic A2AR in st-A2AR KO mice was also consistent with the normal level of A2AR binding density in total membranes [9] and synaptosomal membranes (data not shown) of the cerebral cortex of st-A2AR KO mice. Together, these data demonstrate that A2AR in glutamatergic terminals of the striatum were selectively preserved in st-A2AR KO mice but abolished in fb-A2AR KO mice.

Bottom Line: The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75.Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75.We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and glutamatergic signaling.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Lab, Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Adenosine A2A receptors (A2AR) are located postsynaptically in striatopallidal GABAergic neurons, antagonizing dopamine D2 receptor functions, and are also located presynaptically at corticostriatal terminals, facilitating glutamate release. To address the hypothesis that these two A2AR populations differently control the action of psychostimulants, we characterized A2AR modulation of cocaine-induced effects at the level of DARPP-32 phosphorylation at Thr-34 and Thr-75, c-Fos expression, and psychomotor activity using two lines of cell-type selective A2AR knockout (KO) mice with selective A2AR deletion in GABAergic neurons (striatum-A2AR-KO mice), or with A2AR deletion in both striatal GABAergic neurons and projecting cortical glutamatergic neurons (forebrain-A2AR-KO mice). We demonstrated that striatum-A2AR KO mice lacked A2ARs exclusively in striatal GABAergic terminals whereas forebrain-A2AR KO mice lacked A2ARs in both striatal GABAergic and glutamatergic terminals leading to a blunted A2AR-mediated facilitation of synaptosomal glutamate release. The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75. Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75. This distinct modulation of DARPP-32 phosphorylation was associated with opposite responses to cocaine-induced striatal c-Fos expression and psychomotor activity in striatum-A2AR KO (enhanced) and forebrain-A2AR KO mice (reduced). Thus, A2ARs in glutamatergic corticostriatal terminals and in GABAergic striatal neurons modulate the action of psychostimulants and DARPP-32 phosphorylation in opposite ways. We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and glutamatergic signaling.

Show MeSH
Related in: MedlinePlus