Limits...
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 integrative model of interactions of the different partners involved in the Ca2+-response modulation in the CCB and the medial lobe. In the CCB: the conditional stimulus (e.g., an olfactory stimulus) triggers nicotinic inputs, which activate the nAchR located on the KCs of the MBs, allowing the Ca2+-entry. Calcium binds to CaM and subsequently activates the production of cAMP by RUT. In agreement with the coincidence detector model, in certain conditions, e.g., when the conditional stimulus is simultaneously applied with an unconditional stimulus (e.g., a nociceptive electric shock), the dopaminergic receptors could costimulate the rut-AC to increase further the cAMP level through Gαs. The resulting increase in cAMP stimulates the PKA as well as the CNGs (CNGC and CNGL), which both participate in amplifying the Ca2+-entry. At the same time, the PKA allows the Ca2+-entry and/or the persistence of the Ca2+-entry, likely by affecting the repolarisation of the cells, possibly through the K+-channels. In parallel and simultaneously, the Ca2+-entry modifies the voltage of the cells that allows the voltage-gated calcium channels (VGCC) to also participate to the Ca2+-entry. Altogether, these activities trigger actions potentials (APs) that propagate to the lobes. In the medial lobes: at the axon terminals, the APs open the VGCC, allowing Ca2+-entry. This Ca2+ stimulates the CaM. In parallel or simultaneously (as for instance in certain environmental conditions), a neuromodulator (e.g., dopamine, octopamine) activates a metabotropic receptor, which stimulates a G-protein, and then stimulates the RUT to increase the cAMP. Then, the cAMP stimulates the CNGs (and more likely the CNGL; as suggested by our results in Fig. 6F,H,J). Moreover, according to our results (knocking-down the CaM and handling the cAMP level; Fig. 4), we hypothesize that the CaM might act as an inhibitor of the cAMP-stimulation of the CNGL (red line). This could be achieved either through the CaMKII or CASK, since both of them have been implicated in learning and memory, or directly by the CaM on the CNGL (since in some organisms, certain CNGs have been reported to be sensitive to CaM; Kaupp and Seifert, 2002). These successive events lead to the fine tuning of the Ca2+-level that mobilize the synaptic vesicles and the output. This hypothetical concomitant inhibition by the CaM and the cAMP on the CNGL could represent a coincidence detector.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4596083&req=5

Figure 8: Schematic integrative model of interactions of the different partners involved in the Ca2+-response modulation in the CCB and the medial lobe. In the CCB: the conditional stimulus (e.g., an olfactory stimulus) triggers nicotinic inputs, which activate the nAchR located on the KCs of the MBs, allowing the Ca2+-entry. Calcium binds to CaM and subsequently activates the production of cAMP by RUT. In agreement with the coincidence detector model, in certain conditions, e.g., when the conditional stimulus is simultaneously applied with an unconditional stimulus (e.g., a nociceptive electric shock), the dopaminergic receptors could costimulate the rut-AC to increase further the cAMP level through Gαs. The resulting increase in cAMP stimulates the PKA as well as the CNGs (CNGC and CNGL), which both participate in amplifying the Ca2+-entry. At the same time, the PKA allows the Ca2+-entry and/or the persistence of the Ca2+-entry, likely by affecting the repolarisation of the cells, possibly through the K+-channels. In parallel and simultaneously, the Ca2+-entry modifies the voltage of the cells that allows the voltage-gated calcium channels (VGCC) to also participate to the Ca2+-entry. Altogether, these activities trigger actions potentials (APs) that propagate to the lobes. In the medial lobes: at the axon terminals, the APs open the VGCC, allowing Ca2+-entry. This Ca2+ stimulates the CaM. In parallel or simultaneously (as for instance in certain environmental conditions), a neuromodulator (e.g., dopamine, octopamine) activates a metabotropic receptor, which stimulates a G-protein, and then stimulates the RUT to increase the cAMP. Then, the cAMP stimulates the CNGs (and more likely the CNGL; as suggested by our results in Fig. 6F,H,J). Moreover, according to our results (knocking-down the CaM and handling the cAMP level; Fig. 4), we hypothesize that the CaM might act as an inhibitor of the cAMP-stimulation of the CNGL (red line). This could be achieved either through the CaMKII or CASK, since both of them have been implicated in learning and memory, or directly by the CaM on the CNGL (since in some organisms, certain CNGs have been reported to be sensitive to CaM; Kaupp and Seifert, 2002). These successive events lead to the fine tuning of the Ca2+-level that mobilize the synaptic vesicles and the output. This hypothetical concomitant inhibition by the CaM and the cAMP on the CNGL could represent a coincidence detector.

Mentions: However, the results obtained from the CaM knockdown (CaM-RNAi) combined to either the forskolin or the IBMX or dnc-RNAi (Fig. 4C−E), which all yield a clear dissociated effect between the CCB and the lobes, could suggest a second alternative—for instance, the implication of intermediate partners. Indeed, the effect of the CaM knockdown in the CCB, which seems to be compensated solely by the direct stimulation of the rut-AC (forskolin), is consistent with the canonical model involving CaM directly in the rut stimulation. However, the unexpected striking decreased Ca2+-response in the ML due to the increase of the cAMP (IBMX or dnc-RNAi) combined with CaM knockdown, suggesting that another partner, hypothetically coregulated by cAMP (directly or through PKA) and/or CaM (directly or through CaMKII and/or CASK), could play a role in the ML regulation by inhibiting the Ca2+-response when cAMP is increased in absence of the CaM. Interestingly, the Ca2+-response of the forskolin in the CaM-RNAi (Fig. 4C) resembles the effect of the forskolin, Gαs, and 8-Br-cAMP in the cngl-RNAi context (Fig. 6F,H,J), suggesting that CNGL could be a putative target. Ca2+/calmodulin modulation of different CNGs has also been already reported in other systems as olfactory and visual systems (Trudeau and Zagotta, 2003). Therefore, CASK and/or CaMKII are good candidates for these putative intermediate partners (see Fig. 8 for a schematic model) since both of them have been reported to be involved in learning and memory formation (Malik et al., 2013), as well as in calcium signaling in Drosophila larvae (Gillespie and Hodge, 2013).


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 integrative model of interactions of the different partners involved in the Ca2+-response modulation in the CCB and the medial lobe. In the CCB: the conditional stimulus (e.g., an olfactory stimulus) triggers nicotinic inputs, which activate the nAchR located on the KCs of the MBs, allowing the Ca2+-entry. Calcium binds to CaM and subsequently activates the production of cAMP by RUT. In agreement with the coincidence detector model, in certain conditions, e.g., when the conditional stimulus is simultaneously applied with an unconditional stimulus (e.g., a nociceptive electric shock), the dopaminergic receptors could costimulate the rut-AC to increase further the cAMP level through Gαs. The resulting increase in cAMP stimulates the PKA as well as the CNGs (CNGC and CNGL), which both participate in amplifying the Ca2+-entry. At the same time, the PKA allows the Ca2+-entry and/or the persistence of the Ca2+-entry, likely by affecting the repolarisation of the cells, possibly through the K+-channels. In parallel and simultaneously, the Ca2+-entry modifies the voltage of the cells that allows the voltage-gated calcium channels (VGCC) to also participate to the Ca2+-entry. Altogether, these activities trigger actions potentials (APs) that propagate to the lobes. In the medial lobes: at the axon terminals, the APs open the VGCC, allowing Ca2+-entry. This Ca2+ stimulates the CaM. In parallel or simultaneously (as for instance in certain environmental conditions), a neuromodulator (e.g., dopamine, octopamine) activates a metabotropic receptor, which stimulates a G-protein, and then stimulates the RUT to increase the cAMP. Then, the cAMP stimulates the CNGs (and more likely the CNGL; as suggested by our results in Fig. 6F,H,J). Moreover, according to our results (knocking-down the CaM and handling the cAMP level; Fig. 4), we hypothesize that the CaM might act as an inhibitor of the cAMP-stimulation of the CNGL (red line). This could be achieved either through the CaMKII or CASK, since both of them have been implicated in learning and memory, or directly by the CaM on the CNGL (since in some organisms, certain CNGs have been reported to be sensitive to CaM; Kaupp and Seifert, 2002). These successive events lead to the fine tuning of the Ca2+-level that mobilize the synaptic vesicles and the output. This hypothetical concomitant inhibition by the CaM and the cAMP on the CNGL could represent a coincidence detector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Schematic integrative model of interactions of the different partners involved in the Ca2+-response modulation in the CCB and the medial lobe. In the CCB: the conditional stimulus (e.g., an olfactory stimulus) triggers nicotinic inputs, which activate the nAchR located on the KCs of the MBs, allowing the Ca2+-entry. Calcium binds to CaM and subsequently activates the production of cAMP by RUT. In agreement with the coincidence detector model, in certain conditions, e.g., when the conditional stimulus is simultaneously applied with an unconditional stimulus (e.g., a nociceptive electric shock), the dopaminergic receptors could costimulate the rut-AC to increase further the cAMP level through Gαs. The resulting increase in cAMP stimulates the PKA as well as the CNGs (CNGC and CNGL), which both participate in amplifying the Ca2+-entry. At the same time, the PKA allows the Ca2+-entry and/or the persistence of the Ca2+-entry, likely by affecting the repolarisation of the cells, possibly through the K+-channels. In parallel and simultaneously, the Ca2+-entry modifies the voltage of the cells that allows the voltage-gated calcium channels (VGCC) to also participate to the Ca2+-entry. Altogether, these activities trigger actions potentials (APs) that propagate to the lobes. In the medial lobes: at the axon terminals, the APs open the VGCC, allowing Ca2+-entry. This Ca2+ stimulates the CaM. In parallel or simultaneously (as for instance in certain environmental conditions), a neuromodulator (e.g., dopamine, octopamine) activates a metabotropic receptor, which stimulates a G-protein, and then stimulates the RUT to increase the cAMP. Then, the cAMP stimulates the CNGs (and more likely the CNGL; as suggested by our results in Fig. 6F,H,J). Moreover, according to our results (knocking-down the CaM and handling the cAMP level; Fig. 4), we hypothesize that the CaM might act as an inhibitor of the cAMP-stimulation of the CNGL (red line). This could be achieved either through the CaMKII or CASK, since both of them have been implicated in learning and memory, or directly by the CaM on the CNGL (since in some organisms, certain CNGs have been reported to be sensitive to CaM; Kaupp and Seifert, 2002). These successive events lead to the fine tuning of the Ca2+-level that mobilize the synaptic vesicles and the output. This hypothetical concomitant inhibition by the CaM and the cAMP on the CNGL could represent a coincidence detector.
Mentions: However, the results obtained from the CaM knockdown (CaM-RNAi) combined to either the forskolin or the IBMX or dnc-RNAi (Fig. 4C−E), which all yield a clear dissociated effect between the CCB and the lobes, could suggest a second alternative—for instance, the implication of intermediate partners. Indeed, the effect of the CaM knockdown in the CCB, which seems to be compensated solely by the direct stimulation of the rut-AC (forskolin), is consistent with the canonical model involving CaM directly in the rut stimulation. However, the unexpected striking decreased Ca2+-response in the ML due to the increase of the cAMP (IBMX or dnc-RNAi) combined with CaM knockdown, suggesting that another partner, hypothetically coregulated by cAMP (directly or through PKA) and/or CaM (directly or through CaMKII and/or CASK), could play a role in the ML regulation by inhibiting the Ca2+-response when cAMP is increased in absence of the CaM. Interestingly, the Ca2+-response of the forskolin in the CaM-RNAi (Fig. 4C) resembles the effect of the forskolin, Gαs, and 8-Br-cAMP in the cngl-RNAi context (Fig. 6F,H,J), suggesting that CNGL could be a putative target. Ca2+/calmodulin modulation of different CNGs has also been already reported in other systems as olfactory and visual systems (Trudeau and Zagotta, 2003). Therefore, CASK and/or CaMKII are good candidates for these putative intermediate partners (see Fig. 8 for a schematic model) since both of them have been reported to be involved in learning and memory formation (Malik et al., 2013), as well as in calcium signaling in Drosophila larvae (Gillespie and Hodge, 2013).

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