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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

Schematic view of the setup and a representative nicotine-evoked Ca2+-response in the MBs of a control fly. A, Recording setup. The head capsule of a living fly is opened and the brain is bathed in Ringer’s solution, into which the agonist or antagonist is applied. B, Fluorescent image, taken with a Dim+Fluorescent light, of a 4-d-old female control fly (GFP-aequorin/CS; OK107/CS) after preparation and dissection. C, Fluorescent image of the MBs taken at the beginning of the experiment and used as the reference image. Light emission was quantified from the blue and red circles, which represent the CCB and the ML ROIs, respectively (scale bar, 50 μm). D, Bioluminescence image (accumulation time: 120 s) of the nicotine-evoked response in a typical control fly. E, Bioluminescent Ca2+-activity profile in MBs, evoked by nicotine (n = 15). Values are mean ± SEM. F, Six sequential bioluminescence images from t = −10 s to t = 50 s (accumulation time: 10 s) of the nicotine-evoked response. G, Decomposition image of the CCB showing that the first component corresponds to the response in the calyx (dendrites, ROI circled in green in H), while the second corresponds to that of the cell-bodies (ROI circled in orange in H). Because of the recording angle, the response in the calyx, unavoidably, partially overlaps with the response in the cell bodies. H, Accumulated (10 s) bioluminescence image of the nicotine-evoked response corresponding to each ROI, separately. Because it is not possible to perfectly separate the response from the two ROIs, we use a single ROI comprised of both of them: the CCB complex (ROI circled in blue). For the medial lobes (red circle in C), again here, since we privileged the overall view of the MBs, this approach did not permit us to separate the response of the various sublobes, such as β, β’, or γ.
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Figure 1: Schematic view of the setup and a representative nicotine-evoked Ca2+-response in the MBs of a control fly. A, Recording setup. The head capsule of a living fly is opened and the brain is bathed in Ringer’s solution, into which the agonist or antagonist is applied. B, Fluorescent image, taken with a Dim+Fluorescent light, of a 4-d-old female control fly (GFP-aequorin/CS; OK107/CS) after preparation and dissection. C, Fluorescent image of the MBs taken at the beginning of the experiment and used as the reference image. Light emission was quantified from the blue and red circles, which represent the CCB and the ML ROIs, respectively (scale bar, 50 μm). D, Bioluminescence image (accumulation time: 120 s) of the nicotine-evoked response in a typical control fly. E, Bioluminescent Ca2+-activity profile in MBs, evoked by nicotine (n = 15). Values are mean ± SEM. F, Six sequential bioluminescence images from t = −10 s to t = 50 s (accumulation time: 10 s) of the nicotine-evoked response. G, Decomposition image of the CCB showing that the first component corresponds to the response in the calyx (dendrites, ROI circled in green in H), while the second corresponds to that of the cell-bodies (ROI circled in orange in H). Because of the recording angle, the response in the calyx, unavoidably, partially overlaps with the response in the cell bodies. H, Accumulated (10 s) bioluminescence image of the nicotine-evoked response corresponding to each ROI, separately. Because it is not possible to perfectly separate the response from the two ROIs, we use a single ROI comprised of both of them: the CCB complex (ROI circled in blue). For the medial lobes (red circle in C), again here, since we privileged the overall view of the MBs, this approach did not permit us to separate the response of the various sublobes, such as β, β’, or γ.

Mentions: We used a 20× objective, which allowed visualisation of the entire MB at once, and recorded responses from the CCB and various MB lobes (Fig. 1A−C), which could be subdivided into the vertical lobe, comprised of the α/α’ lobes, and the medial lobes, comprised of the β/β’ and γ lobes. In the absence of any stimulus, we observed neither basal nor oscillatory Ca2+-activity in the KCs (the constitutive neurons of the MBs). A 1 min application of nicotine (25 μM, at 2 ml/min) evoked a typical response pattern in the MBs. The response started in the CCB and propagated into the axonal projections at the level of the MB lobes (Fig. 1D−F). A typical nicotine-evoked Ca2+-response was composed of two distinct phases in the CCB, and only one phase in the MB lobes. The CCB response first showed a rapid exponential activity increase (0 s corresponds to the beginning of the response), and peaked at approximately 9 s (Fig. 1E). This first phase reached ∼2200 photons/s (ph/s), the signal then decreased slightly for ∼2 s, and rose again to give a second lower peak of ∼1800 ph/s, ∼15 s after the first response started. The responses finally decreased slowly, and terminated after ∼80 s. To simplify, the response can be summarized into two components, which are defined by the first and the second peak. In addition, the use of different angles of view to observe the MB permitted the identification of substructures associated with both response components. The first component corresponds to the response in the calyx (Fig. 1H, green ROI), while the second component, which occurs slightly after, corresponds the response in the cell bodies of the KCs (Fig. 1H, orange ROI). Indeed, a refinement of the two ROIs, which was possible on few flies according to their precise angles of view, allowed spatiotemporal separation of these two components of the response (Fig. 1G). However, as the two components partly overlap in the majority of the flies imaged, it made it difficult to precisely and systematically separate the two components and to define their individual durations. Consequently, only the overall response of the CCB and duration were taken into account in this study.


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)

Schematic view of the setup and a representative nicotine-evoked Ca2+-response in the MBs of a control fly. A, Recording setup. The head capsule of a living fly is opened and the brain is bathed in Ringer’s solution, into which the agonist or antagonist is applied. B, Fluorescent image, taken with a Dim+Fluorescent light, of a 4-d-old female control fly (GFP-aequorin/CS; OK107/CS) after preparation and dissection. C, Fluorescent image of the MBs taken at the beginning of the experiment and used as the reference image. Light emission was quantified from the blue and red circles, which represent the CCB and the ML ROIs, respectively (scale bar, 50 μm). D, Bioluminescence image (accumulation time: 120 s) of the nicotine-evoked response in a typical control fly. E, Bioluminescent Ca2+-activity profile in MBs, evoked by nicotine (n = 15). Values are mean ± SEM. F, Six sequential bioluminescence images from t = −10 s to t = 50 s (accumulation time: 10 s) of the nicotine-evoked response. G, Decomposition image of the CCB showing that the first component corresponds to the response in the calyx (dendrites, ROI circled in green in H), while the second corresponds to that of the cell-bodies (ROI circled in orange in H). Because of the recording angle, the response in the calyx, unavoidably, partially overlaps with the response in the cell bodies. H, Accumulated (10 s) bioluminescence image of the nicotine-evoked response corresponding to each ROI, separately. Because it is not possible to perfectly separate the response from the two ROIs, we use a single ROI comprised of both of them: the CCB complex (ROI circled in blue). For the medial lobes (red circle in C), again here, since we privileged the overall view of the MBs, this approach did not permit us to separate the response of the various sublobes, such as β, β’, or γ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596083&req=5

Figure 1: Schematic view of the setup and a representative nicotine-evoked Ca2+-response in the MBs of a control fly. A, Recording setup. The head capsule of a living fly is opened and the brain is bathed in Ringer’s solution, into which the agonist or antagonist is applied. B, Fluorescent image, taken with a Dim+Fluorescent light, of a 4-d-old female control fly (GFP-aequorin/CS; OK107/CS) after preparation and dissection. C, Fluorescent image of the MBs taken at the beginning of the experiment and used as the reference image. Light emission was quantified from the blue and red circles, which represent the CCB and the ML ROIs, respectively (scale bar, 50 μm). D, Bioluminescence image (accumulation time: 120 s) of the nicotine-evoked response in a typical control fly. E, Bioluminescent Ca2+-activity profile in MBs, evoked by nicotine (n = 15). Values are mean ± SEM. F, Six sequential bioluminescence images from t = −10 s to t = 50 s (accumulation time: 10 s) of the nicotine-evoked response. G, Decomposition image of the CCB showing that the first component corresponds to the response in the calyx (dendrites, ROI circled in green in H), while the second corresponds to that of the cell-bodies (ROI circled in orange in H). Because of the recording angle, the response in the calyx, unavoidably, partially overlaps with the response in the cell bodies. H, Accumulated (10 s) bioluminescence image of the nicotine-evoked response corresponding to each ROI, separately. Because it is not possible to perfectly separate the response from the two ROIs, we use a single ROI comprised of both of them: the CCB complex (ROI circled in blue). For the medial lobes (red circle in C), again here, since we privileged the overall view of the MBs, this approach did not permit us to separate the response of the various sublobes, such as β, β’, or γ.
Mentions: We used a 20× objective, which allowed visualisation of the entire MB at once, and recorded responses from the CCB and various MB lobes (Fig. 1A−C), which could be subdivided into the vertical lobe, comprised of the α/α’ lobes, and the medial lobes, comprised of the β/β’ and γ lobes. In the absence of any stimulus, we observed neither basal nor oscillatory Ca2+-activity in the KCs (the constitutive neurons of the MBs). A 1 min application of nicotine (25 μM, at 2 ml/min) evoked a typical response pattern in the MBs. The response started in the CCB and propagated into the axonal projections at the level of the MB lobes (Fig. 1D−F). A typical nicotine-evoked Ca2+-response was composed of two distinct phases in the CCB, and only one phase in the MB lobes. The CCB response first showed a rapid exponential activity increase (0 s corresponds to the beginning of the response), and peaked at approximately 9 s (Fig. 1E). This first phase reached ∼2200 photons/s (ph/s), the signal then decreased slightly for ∼2 s, and rose again to give a second lower peak of ∼1800 ph/s, ∼15 s after the first response started. The responses finally decreased slowly, and terminated after ∼80 s. To simplify, the response can be summarized into two components, which are defined by the first and the second peak. In addition, the use of different angles of view to observe the MB permitted the identification of substructures associated with both response components. The first component corresponds to the response in the calyx (Fig. 1H, green ROI), while the second component, which occurs slightly after, corresponds the response in the cell bodies of the KCs (Fig. 1H, orange ROI). Indeed, a refinement of the two ROIs, which was possible on few flies according to their precise angles of view, allowed spatiotemporal separation of these two components of the response (Fig. 1G). However, as the two components partly overlap in the majority of the flies imaged, it made it difficult to precisely and systematically separate the two components and to define their individual durations. Consequently, only the overall response of the CCB and duration were taken into account in this study.

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