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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.


PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nm). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, 1 µm), or SKF-38393, a D1-like receptor agonist (SKF, 1 µm). The thick black line represents the average of all the traces in a group. FSK (13 µm) and IBMX (200 µm) were applied at the end of the recording to determine the maximal response. B, The same experiment was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: dose effect, F(6,54) = 40.91, p < 10−4; D1/D2 effect, F(1,54) = 2.56, p = 0.115; dose × D1/D2 interaction, F(6,54) = 0.625, p = 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose effect, F(6,38) = 28.31, p < 10−4; D1/D2 effect, F(1,38) = 143.73, p < 10−4; dose × D1/D2 interaction, F(6,38) = 9.23, p < 10−4 Bonferroni’s post hoc test, ***p < 0.001 .
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Figure 1: PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nm). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, 1 µm), or SKF-38393, a D1-like receptor agonist (SKF, 1 µm). The thick black line represents the average of all the traces in a group. FSK (13 µm) and IBMX (200 µm) were applied at the end of the recording to determine the maximal response. B, The same experiment was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: dose effect, F(6,54) = 40.91, p < 10−4; D1/D2 effect, F(1,54) = 2.56, p = 0.115; dose × D1/D2 interaction, F(6,54) = 0.625, p = 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose effect, F(6,38) = 28.31, p < 10−4; D1/D2 effect, F(1,38) = 143.73, p < 10−4; dose × D1/D2 interaction, F(6,38) = 9.23, p < 10−4 Bonferroni’s post hoc test, ***p < 0.001 .

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)

PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nm). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, 1 µm), or SKF-38393, a D1-like receptor agonist (SKF, 1 µm). The thick black line represents the average of all the traces in a group. FSK (13 µm) and IBMX (200 µm) were applied at the end of the recording to determine the maximal response. B, The same experiment was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: dose effect, F(6,54) = 40.91, p < 10−4; D1/D2 effect, F(1,54) = 2.56, p = 0.115; dose × D1/D2 interaction, F(6,54) = 0.625, p = 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose effect, F(6,38) = 28.31, p < 10−4; D1/D2 effect, F(1,38) = 143.73, p < 10−4; dose × D1/D2 interaction, F(6,38) = 9.23, p < 10−4 Bonferroni’s post hoc test, ***p < 0.001 .
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Figure 1: PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nm). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, 1 µm), or SKF-38393, a D1-like receptor agonist (SKF, 1 µm). The thick black line represents the average of all the traces in a group. FSK (13 µm) and IBMX (200 µm) were applied at the end of the recording to determine the maximal response. B, The same experiment was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: dose effect, F(6,54) = 40.91, p < 10−4; D1/D2 effect, F(1,54) = 2.56, p = 0.115; dose × D1/D2 interaction, F(6,54) = 0.625, p = 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose effect, F(6,38) = 28.31, p < 10−4; D1/D2 effect, F(1,38) = 143.73, p < 10−4; dose × D1/D2 interaction, F(6,38) = 9.23, p < 10−4 Bonferroni’s post hoc test, ***p < 0.001 .
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.