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PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca(2+)-Response of Drosophila Mushroom Body Neurons(1,2,3).

Pavot P, Carbognin E, Martin JR - eNeuro (2015)

Bottom Line: Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG).Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC.Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Neurosciences Paris-Saclay (Neuro-PSI), UMR-9197, CNRS/Université Paris Sud , 91198, Gif-sur-Yvette, France.

ABSTRACT
The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca(2+)-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca(2+)-activity continuously, over a long time period, and characterized the nicotine-evoked Ca(2+)-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca(2+)-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

No MeSH data available.


Related in: MedlinePlus

Modulation of nicotine-evoked transient Ca2+-response related to the cAMP pathway through rut. A−D, Bioluminescent Ca2+-activity profile in MBs evoked by nicotine with downregulated cAMP production in rut1 (n = 6), rut2080 (continued in page 7). (n = 7), rut-RNAi(1) (n = 8), and rut-RNAi(2) (n = 6). E−H, Bioluminescent Ca2+-activity evoked by nicotine with upregulated cAMP production in UAS-rut (n = 8), UAS-Gαs* (n = 7), flies incubated 10 min with forskolin (13 μM; n = 6), and flies incubated 10 min with 8Br-cAMP (200 μM; n = 7). I, J, Bioluminescent nicotine-evoked Ca2+-activity with different genetic background controls in Control-RNAi(1) (VDRC-60100: n = 17; I) and in Control-RNAi(2) (VDRC-60000: n = 15; J) as well as in Control-RNAi(2), incubated 10 min with ethanol 1/200 (n = 11; J). Values are mean ± SEM. K, Bioluminescent image (accumulation time: 120 s) of the nicotinic Ca2+-response of a typical fly for each genotype (except for the other controls presented in I and J). L, M, Total number of photons during the nicotine response in the CCB (L) and in the ML (M). N, O, Total duration of the response in the CCB (N) and in the medial lobe (O). Values are mean ± SEM. Statistics: A−D, One-way ANOVA was followed by a planned comparison of the predicted means to compare the levels of the selected effect using the Benjamini-Hochberg's test with rank transformation: *p < 0.05; **p < 0.01; ***p < 0.001 (for complete statistics, see Tables 1 and 2).
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Figure 2: Modulation of nicotine-evoked transient Ca2+-response related to the cAMP pathway through rut. A−D, Bioluminescent Ca2+-activity profile in MBs evoked by nicotine with downregulated cAMP production in rut1 (n = 6), rut2080 (continued in page 7). (n = 7), rut-RNAi(1) (n = 8), and rut-RNAi(2) (n = 6). E−H, Bioluminescent Ca2+-activity evoked by nicotine with upregulated cAMP production in UAS-rut (n = 8), UAS-Gαs* (n = 7), flies incubated 10 min with forskolin (13 μM; n = 6), and flies incubated 10 min with 8Br-cAMP (200 μM; n = 7). I, J, Bioluminescent nicotine-evoked Ca2+-activity with different genetic background controls in Control-RNAi(1) (VDRC-60100: n = 17; I) and in Control-RNAi(2) (VDRC-60000: n = 15; J) as well as in Control-RNAi(2), incubated 10 min with ethanol 1/200 (n = 11; J). Values are mean ± SEM. K, Bioluminescent image (accumulation time: 120 s) of the nicotinic Ca2+-response of a typical fly for each genotype (except for the other controls presented in I and J). L, M, Total number of photons during the nicotine response in the CCB (L) and in the ML (M). N, O, Total duration of the response in the CCB (N) and in the medial lobe (O). Values are mean ± SEM. Statistics: A−D, One-way ANOVA was followed by a planned comparison of the predicted means to compare the levels of the selected effect using the Benjamini-Hochberg's test with rank transformation: *p < 0.05; **p < 0.01; ***p < 0.001 (for complete statistics, see Tables 1 and 2).

Mentions: Similarly, the spatial resolution obtained at the level of the MB lobes did not allow us to precisely discriminate different subneuronal populations from each other. Therefore, the α/α’ lobes are considered altogether as the vertical lobe (VL), while the β/β’ and γ lobes are considered altogether as the medial lobes (ML) in this study. Moreover, due to the position of the fly’s head and the recording angle, the VLs partly overlapped with the peduncles of the MBs. Thus, in order to avoid any bias in subsequent analysis, we only quantified the response in the CCB and ML. In summary, the first component of the response corresponds to the calyx (dendritic branches), whereas the second component corresponds to the cell bodies (Fig. 1E). The response in the ML (Fig. 1E, red curve) was delayed compared to the CCB response, and was composed of a single peak of approximately 1100 ph/s, which occurred roughly 10 s after response initiation. The ML response lasted for about 55 s in total. We also quantified the total number of emitted photons for the response in the CCB and ML. The TP average was ∼39000 photons from the CCB and ∼13000 from the ML (see Fig. 3). Finally, to confirm that this robust Ca2+-response does not significantly vary with genetic background, results were obtained with additional control lines (VDRC-GD-60000 and VDRC-KK-61000), which were recorded and then shown to share the same characteristics as the CS trans-heterozygotes flies (Fig. 2I−J,L−O, blue bars).


PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca(2+)-Response of Drosophila Mushroom Body Neurons(1,2,3).

Pavot P, Carbognin E, Martin JR - eNeuro (2015)

Modulation of nicotine-evoked transient Ca2+-response related to the cAMP pathway through rut. A−D, Bioluminescent Ca2+-activity profile in MBs evoked by nicotine with downregulated cAMP production in rut1 (n = 6), rut2080 (continued in page 7). (n = 7), rut-RNAi(1) (n = 8), and rut-RNAi(2) (n = 6). E−H, Bioluminescent Ca2+-activity evoked by nicotine with upregulated cAMP production in UAS-rut (n = 8), UAS-Gαs* (n = 7), flies incubated 10 min with forskolin (13 μM; n = 6), and flies incubated 10 min with 8Br-cAMP (200 μM; n = 7). I, J, Bioluminescent nicotine-evoked Ca2+-activity with different genetic background controls in Control-RNAi(1) (VDRC-60100: n = 17; I) and in Control-RNAi(2) (VDRC-60000: n = 15; J) as well as in Control-RNAi(2), incubated 10 min with ethanol 1/200 (n = 11; J). Values are mean ± SEM. K, Bioluminescent image (accumulation time: 120 s) of the nicotinic Ca2+-response of a typical fly for each genotype (except for the other controls presented in I and J). L, M, Total number of photons during the nicotine response in the CCB (L) and in the ML (M). N, O, Total duration of the response in the CCB (N) and in the medial lobe (O). Values are mean ± SEM. Statistics: A−D, One-way ANOVA was followed by a planned comparison of the predicted means to compare the levels of the selected effect using the Benjamini-Hochberg's test with rank transformation: *p < 0.05; **p < 0.01; ***p < 0.001 (for complete statistics, see Tables 1 and 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Modulation of nicotine-evoked transient Ca2+-response related to the cAMP pathway through rut. A−D, Bioluminescent Ca2+-activity profile in MBs evoked by nicotine with downregulated cAMP production in rut1 (n = 6), rut2080 (continued in page 7). (n = 7), rut-RNAi(1) (n = 8), and rut-RNAi(2) (n = 6). E−H, Bioluminescent Ca2+-activity evoked by nicotine with upregulated cAMP production in UAS-rut (n = 8), UAS-Gαs* (n = 7), flies incubated 10 min with forskolin (13 μM; n = 6), and flies incubated 10 min with 8Br-cAMP (200 μM; n = 7). I, J, Bioluminescent nicotine-evoked Ca2+-activity with different genetic background controls in Control-RNAi(1) (VDRC-60100: n = 17; I) and in Control-RNAi(2) (VDRC-60000: n = 15; J) as well as in Control-RNAi(2), incubated 10 min with ethanol 1/200 (n = 11; J). Values are mean ± SEM. K, Bioluminescent image (accumulation time: 120 s) of the nicotinic Ca2+-response of a typical fly for each genotype (except for the other controls presented in I and J). L, M, Total number of photons during the nicotine response in the CCB (L) and in the ML (M). N, O, Total duration of the response in the CCB (N) and in the medial lobe (O). Values are mean ± SEM. Statistics: A−D, One-way ANOVA was followed by a planned comparison of the predicted means to compare the levels of the selected effect using the Benjamini-Hochberg's test with rank transformation: *p < 0.05; **p < 0.01; ***p < 0.001 (for complete statistics, see Tables 1 and 2).
Mentions: Similarly, the spatial resolution obtained at the level of the MB lobes did not allow us to precisely discriminate different subneuronal populations from each other. Therefore, the α/α’ lobes are considered altogether as the vertical lobe (VL), while the β/β’ and γ lobes are considered altogether as the medial lobes (ML) in this study. Moreover, due to the position of the fly’s head and the recording angle, the VLs partly overlapped with the peduncles of the MBs. Thus, in order to avoid any bias in subsequent analysis, we only quantified the response in the CCB and ML. In summary, the first component of the response corresponds to the calyx (dendritic branches), whereas the second component corresponds to the cell bodies (Fig. 1E). The response in the ML (Fig. 1E, red curve) was delayed compared to the CCB response, and was composed of a single peak of approximately 1100 ph/s, which occurred roughly 10 s after response initiation. The ML response lasted for about 55 s in total. We also quantified the total number of emitted photons for the response in the CCB and ML. The TP average was ∼39000 photons from the CCB and ∼13000 from the ML (see Fig. 3). Finally, to confirm that this robust Ca2+-response does not significantly vary with genetic background, results were obtained with additional control lines (VDRC-GD-60000 and VDRC-KK-61000), which were recorded and then shown to share the same characteristics as the CS trans-heterozygotes flies (Fig. 2I−J,L−O, blue bars).

Bottom Line: Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG).Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC.Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Neurosciences Paris-Saclay (Neuro-PSI), UMR-9197, CNRS/Université Paris Sud , 91198, Gif-sur-Yvette, France.

ABSTRACT
The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca(2+)-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca(2+)-activity continuously, over a long time period, and characterized the nicotine-evoked Ca(2+)-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca(2+)-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

No MeSH data available.


Related in: MedlinePlus