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Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32(1,2,3).

Polito M, Guiot E, Gangarossa G, Longueville S, Doulazmi M, Valjent E, Hervé D, Girault JA, Paupardin-Tritsch D, Castro LR, Vincent P - eNeuro (2015)

Bottom Line: PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8256 "Biological Adaptation and Ageing", Institut de Biologie Paris-Seine (IBPS) , F-75005 Paris, France ; Université Pierre et Marie Curie (UPMC, Paris 6), Sorbonne Universités , Paris, F-75005, France.

ABSTRACT
Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

No MeSH data available.


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A, Activation of D2 dopamine receptors suppressed the effect of PDE10A inhibition on AKAR3 ratio. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in a group. Bath application of the agonist of dopamine D2 receptors quinpirole (1 µm) reversed the response to PQ-10 (100 nm). B, D2 receptor activation prevented the response to PDE10A inhibition: the effect of PQ-10 was measured in the presence of the D2 agonist quinpirole (1 µm). No statistically significant difference (p > 0.05) was found between D1 and D2 MSNs (n = 5). The effect of PQ-10 is displayed for comparison on the left (same data as in Fig. 4E). C, D, Other PDE10A inhibitors also increased the AKAR3 ratio preferentially in D2 MSNs: MP-10 (C; 100 nm, n = 9) and papaverine (D; 1 µm, n = 5) both increased the AKAR3 ratio selectively in D2 MSNs. E, PQ-10 increased AKAR3 ratio selectively in D2 MSNS even when adenosine A2A receptors were inhibited with 100 µm SCH 58261 (n = 4). B–E, Statistical differences were tested with paired Student’s t test. ***p < 0.001. F, PDE10A inhibition had no effect on cGMP levels measured with the cGMP sensor cygnet2. The NO donor DEANO (100 µm) increased the ratio; after reaching a steady-state level, PQ-10 (1 µm) was added; at the end of the recording, the maximal ratio response was elicited by DEANO plus IBMX (200 µm). G, No difference was measured when comparing the response with DEANO alone and DEANO with PQ-10, while IBMX produced a significant increase. The data expressed as the mean ± SEM were analyzed by repeated-measures one-way ANOVA F(1,5) = 11,224, p < 0.001, n = 6, followed by Bonferroni’s post hoc test: **p < 0.01). A–G, Brain slices were imaged with wide-field microscopy. All data are expressed as the mean ± SEM.
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Figure 3: A, Activation of D2 dopamine receptors suppressed the effect of PDE10A inhibition on AKAR3 ratio. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in a group. Bath application of the agonist of dopamine D2 receptors quinpirole (1 µm) reversed the response to PQ-10 (100 nm). B, D2 receptor activation prevented the response to PDE10A inhibition: the effect of PQ-10 was measured in the presence of the D2 agonist quinpirole (1 µm). No statistically significant difference (p > 0.05) was found between D1 and D2 MSNs (n = 5). The effect of PQ-10 is displayed for comparison on the left (same data as in Fig. 4E). C, D, Other PDE10A inhibitors also increased the AKAR3 ratio preferentially in D2 MSNs: MP-10 (C; 100 nm, n = 9) and papaverine (D; 1 µm, n = 5) both increased the AKAR3 ratio selectively in D2 MSNs. E, PQ-10 increased AKAR3 ratio selectively in D2 MSNS even when adenosine A2A receptors were inhibited with 100 µm SCH 58261 (n = 4). B–E, Statistical differences were tested with paired Student’s t test. ***p < 0.001. F, PDE10A inhibition had no effect on cGMP levels measured with the cGMP sensor cygnet2. The NO donor DEANO (100 µm) increased the ratio; after reaching a steady-state level, PQ-10 (1 µm) was added; at the end of the recording, the maximal ratio response was elicited by DEANO plus IBMX (200 µm). G, No difference was measured when comparing the response with DEANO alone and DEANO with PQ-10, while IBMX produced a significant increase. The data expressed as the mean ± SEM were analyzed by repeated-measures one-way ANOVA F(1,5) = 11,224, p < 0.001, n = 6, followed by Bonferroni’s post hoc test: **p < 0.01). A–G, Brain slices were imaged with wide-field microscopy. All data are expressed as the mean ± SEM.

Mentions: Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.


Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32(1,2,3).

Polito M, Guiot E, Gangarossa G, Longueville S, Doulazmi M, Valjent E, Hervé D, Girault JA, Paupardin-Tritsch D, Castro LR, Vincent P - eNeuro (2015)

A, Activation of D2 dopamine receptors suppressed the effect of PDE10A inhibition on AKAR3 ratio. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in a group. Bath application of the agonist of dopamine D2 receptors quinpirole (1 µm) reversed the response to PQ-10 (100 nm). B, D2 receptor activation prevented the response to PDE10A inhibition: the effect of PQ-10 was measured in the presence of the D2 agonist quinpirole (1 µm). No statistically significant difference (p > 0.05) was found between D1 and D2 MSNs (n = 5). The effect of PQ-10 is displayed for comparison on the left (same data as in Fig. 4E). C, D, Other PDE10A inhibitors also increased the AKAR3 ratio preferentially in D2 MSNs: MP-10 (C; 100 nm, n = 9) and papaverine (D; 1 µm, n = 5) both increased the AKAR3 ratio selectively in D2 MSNs. E, PQ-10 increased AKAR3 ratio selectively in D2 MSNS even when adenosine A2A receptors were inhibited with 100 µm SCH 58261 (n = 4). B–E, Statistical differences were tested with paired Student’s t test. ***p < 0.001. F, PDE10A inhibition had no effect on cGMP levels measured with the cGMP sensor cygnet2. The NO donor DEANO (100 µm) increased the ratio; after reaching a steady-state level, PQ-10 (1 µm) was added; at the end of the recording, the maximal ratio response was elicited by DEANO plus IBMX (200 µm). G, No difference was measured when comparing the response with DEANO alone and DEANO with PQ-10, while IBMX produced a significant increase. The data expressed as the mean ± SEM were analyzed by repeated-measures one-way ANOVA F(1,5) = 11,224, p < 0.001, n = 6, followed by Bonferroni’s post hoc test: **p < 0.01). A–G, Brain slices were imaged with wide-field microscopy. All data are expressed as the mean ± SEM.
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Figure 3: A, Activation of D2 dopamine receptors suppressed the effect of PDE10A inhibition on AKAR3 ratio. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in a group. Bath application of the agonist of dopamine D2 receptors quinpirole (1 µm) reversed the response to PQ-10 (100 nm). B, D2 receptor activation prevented the response to PDE10A inhibition: the effect of PQ-10 was measured in the presence of the D2 agonist quinpirole (1 µm). No statistically significant difference (p > 0.05) was found between D1 and D2 MSNs (n = 5). The effect of PQ-10 is displayed for comparison on the left (same data as in Fig. 4E). C, D, Other PDE10A inhibitors also increased the AKAR3 ratio preferentially in D2 MSNs: MP-10 (C; 100 nm, n = 9) and papaverine (D; 1 µm, n = 5) both increased the AKAR3 ratio selectively in D2 MSNs. E, PQ-10 increased AKAR3 ratio selectively in D2 MSNS even when adenosine A2A receptors were inhibited with 100 µm SCH 58261 (n = 4). B–E, Statistical differences were tested with paired Student’s t test. ***p < 0.001. F, PDE10A inhibition had no effect on cGMP levels measured with the cGMP sensor cygnet2. The NO donor DEANO (100 µm) increased the ratio; after reaching a steady-state level, PQ-10 (1 µm) was added; at the end of the recording, the maximal ratio response was elicited by DEANO plus IBMX (200 µm). G, No difference was measured when comparing the response with DEANO alone and DEANO with PQ-10, while IBMX produced a significant increase. The data expressed as the mean ± SEM were analyzed by repeated-measures one-way ANOVA F(1,5) = 11,224, p < 0.001, n = 6, followed by Bonferroni’s post hoc test: **p < 0.01). A–G, Brain slices were imaged with wide-field microscopy. All data are expressed as the mean ± SEM.
Mentions: Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.

Bottom Line: PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8256 "Biological Adaptation and Ageing", Institut de Biologie Paris-Seine (IBPS) , F-75005 Paris, France ; Université Pierre et Marie Curie (UPMC, Paris 6), Sorbonne Universités , Paris, F-75005, France.

ABSTRACT
Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

No MeSH data available.


Related in: MedlinePlus