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A single pair of neurons links sleep to memory consolidation in Drosophila melanogaster.

Haynes PR, Christmann BL, Griffith LC - Elife (2015)

Bottom Line: Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition.Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy.These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Volen Center for Complex Systems, National Center for Behavioral Genomics, Brandeis University, Waltham, United States.

ABSTRACT
Sleep promotes memory consolidation in humans and many other species, but the physiological and anatomical relationships between sleep and memory remain unclear. Here, we show the dorsal paired medial (DPM) neurons, which are required for memory consolidation in Drosophila, are sleep-promoting inhibitory neurons. DPMs increase sleep via release of GABA onto wake-promoting mushroom body (MB) α'/β' neurons. Functional imaging demonstrates that DPM activation evokes robust increases in chloride in MB neurons, but is unable to cause detectable increases in calcium or cAMP. Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition. Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy. These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.

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DPM activation has no excitatory effect on the MBs.(A–C) Black bar denotes time of perfusion of 2.5 mM ATP or vehicle. Histograms summarize mean maximum percentage change in fluorescence of respective sensor. (A) Bath-applied ATP is effective at activating DPMs expressing P2X2 receptors. Mean GCaMP3.0 response traces of w-; UAS-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA to 30 s perfusion of 2.5 mM ATP (pink) or vehicle (black). N = 6 for ATP responses, 3 for vehicle responses, p = 0.02 for Mann–Whitney U test, histogram values are 23.7 + 2.1% (pink), 1.2 + 0.7% (black). (B) Mean GCaMP3.0 response traces of w-; lexAop-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA (green), or without UAS-P2X2 transgene (grey), to 30 s perfusion of 2.5 mM ATP or vehicle (black). N = 5 with UAS-P2X2 transgene, 5 without [5, 5], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 1.7 + 0.3% (green), 1.5 + 0.8% (black), 1.2 + 0.5% (grey). (C) Mean EPAC response traces of w-; lexAop-EPAC/NP2721-GAL4; UAS-P2X2/MB247-lexA (orange), or without UAS-P2X2 transgene (grey), to 90 s perfusion of 2.5 mM ATP or vehicle (black). N = [8, 6], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 11.4 + 0.9% (orange), 9.8 + 1.0% (black), 11.4 + 1.1% (grey). (B–C), ROIs were also taken from the vertical lobes and no change in fluorescence was seen (data not shown).DOI:http://dx.doi.org/10.7554/eLife.03868.014
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fig4s1: DPM activation has no excitatory effect on the MBs.(A–C) Black bar denotes time of perfusion of 2.5 mM ATP or vehicle. Histograms summarize mean maximum percentage change in fluorescence of respective sensor. (A) Bath-applied ATP is effective at activating DPMs expressing P2X2 receptors. Mean GCaMP3.0 response traces of w-; UAS-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA to 30 s perfusion of 2.5 mM ATP (pink) or vehicle (black). N = 6 for ATP responses, 3 for vehicle responses, p = 0.02 for Mann–Whitney U test, histogram values are 23.7 + 2.1% (pink), 1.2 + 0.7% (black). (B) Mean GCaMP3.0 response traces of w-; lexAop-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA (green), or without UAS-P2X2 transgene (grey), to 30 s perfusion of 2.5 mM ATP or vehicle (black). N = 5 with UAS-P2X2 transgene, 5 without [5, 5], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 1.7 + 0.3% (green), 1.5 + 0.8% (black), 1.2 + 0.5% (grey). (C) Mean EPAC response traces of w-; lexAop-EPAC/NP2721-GAL4; UAS-P2X2/MB247-lexA (orange), or without UAS-P2X2 transgene (grey), to 90 s perfusion of 2.5 mM ATP or vehicle (black). N = [8, 6], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 11.4 + 0.9% (orange), 9.8 + 1.0% (black), 11.4 + 1.1% (grey). (B–C), ROIs were also taken from the vertical lobes and no change in fluorescence was seen (data not shown).DOI:http://dx.doi.org/10.7554/eLife.03868.014

Mentions: These results suggest that DPM neurons might be inhibitory rather than excitatory, and are inconsistent with a role for DPM neurons in directly enhancing potentiation. To test the sign of the connection, we first used functional imaging techniques to determine if DPM activation could stimulate postsynaptic MB neurons (Figure 4A). We expressed the mammalian ATP-gated P2X2 receptor (Lima and Miesenbock, 2005; Yao et al., 2012) in DPMs using NP2721-GAL4 or c316-GAL4, and activated these cells by applying ATP to dissected adult Drosophila brains. We first confirmed that bath-applied ATP was sufficient to activate the P2X2 receptors in DPMs by co-expressing genetically-encoded fluorescent sensors and using functional imaging to observe changes in fluorescence indicating a response. Using c316-GAL4 to drive UAS-GCaMP3.0 (Tian et al., 2009), UAS-Arclight (Cao et al., 2013), and UAS-Synapto-pHlorin (SpH) (Meisenbock et al., 1998), we found that P2X2-mediated stimulation effectively activated DPM neurons, evoking increases in intracellular calcium, membrane voltage, and vesicle fusion, respectively (Figure 4B). It should be noted that these responses were observed in the DPM projections to the MBs, not the DPM cell bodies, demonstrating that this technique successfully activates the DPMs and causes them to release neurotransmitter from their projections onto downstream targets in the MB neuropil. We also co-expressed P2X2 receptors and UAS-GCaMP3.0 in the DPMs using NP2721-GAL4 to confirm that this technique was effective with the weaker driver (Figure 4—figure supplement 1A).10.7554/eLife.03868.013Figure 4.DPM activation has no excitatory effect on the MBs.


A single pair of neurons links sleep to memory consolidation in Drosophila melanogaster.

Haynes PR, Christmann BL, Griffith LC - Elife (2015)

DPM activation has no excitatory effect on the MBs.(A–C) Black bar denotes time of perfusion of 2.5 mM ATP or vehicle. Histograms summarize mean maximum percentage change in fluorescence of respective sensor. (A) Bath-applied ATP is effective at activating DPMs expressing P2X2 receptors. Mean GCaMP3.0 response traces of w-; UAS-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA to 30 s perfusion of 2.5 mM ATP (pink) or vehicle (black). N = 6 for ATP responses, 3 for vehicle responses, p = 0.02 for Mann–Whitney U test, histogram values are 23.7 + 2.1% (pink), 1.2 + 0.7% (black). (B) Mean GCaMP3.0 response traces of w-; lexAop-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA (green), or without UAS-P2X2 transgene (grey), to 30 s perfusion of 2.5 mM ATP or vehicle (black). N = 5 with UAS-P2X2 transgene, 5 without [5, 5], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 1.7 + 0.3% (green), 1.5 + 0.8% (black), 1.2 + 0.5% (grey). (C) Mean EPAC response traces of w-; lexAop-EPAC/NP2721-GAL4; UAS-P2X2/MB247-lexA (orange), or without UAS-P2X2 transgene (grey), to 90 s perfusion of 2.5 mM ATP or vehicle (black). N = [8, 6], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 11.4 + 0.9% (orange), 9.8 + 1.0% (black), 11.4 + 1.1% (grey). (B–C), ROIs were also taken from the vertical lobes and no change in fluorescence was seen (data not shown).DOI:http://dx.doi.org/10.7554/eLife.03868.014
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fig4s1: DPM activation has no excitatory effect on the MBs.(A–C) Black bar denotes time of perfusion of 2.5 mM ATP or vehicle. Histograms summarize mean maximum percentage change in fluorescence of respective sensor. (A) Bath-applied ATP is effective at activating DPMs expressing P2X2 receptors. Mean GCaMP3.0 response traces of w-; UAS-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA to 30 s perfusion of 2.5 mM ATP (pink) or vehicle (black). N = 6 for ATP responses, 3 for vehicle responses, p = 0.02 for Mann–Whitney U test, histogram values are 23.7 + 2.1% (pink), 1.2 + 0.7% (black). (B) Mean GCaMP3.0 response traces of w-; lexAop-GCaMP3.0/NP2721-GAL4; UAS-P2X2/MB247-lexA (green), or without UAS-P2X2 transgene (grey), to 30 s perfusion of 2.5 mM ATP or vehicle (black). N = 5 with UAS-P2X2 transgene, 5 without [5, 5], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 1.7 + 0.3% (green), 1.5 + 0.8% (black), 1.2 + 0.5% (grey). (C) Mean EPAC response traces of w-; lexAop-EPAC/NP2721-GAL4; UAS-P2X2/MB247-lexA (orange), or without UAS-P2X2 transgene (grey), to 90 s perfusion of 2.5 mM ATP or vehicle (black). N = [8, 6], p > 0.05 for Kruskal–Wallis one-way ANOVA, histogram values are 11.4 + 0.9% (orange), 9.8 + 1.0% (black), 11.4 + 1.1% (grey). (B–C), ROIs were also taken from the vertical lobes and no change in fluorescence was seen (data not shown).DOI:http://dx.doi.org/10.7554/eLife.03868.014
Mentions: These results suggest that DPM neurons might be inhibitory rather than excitatory, and are inconsistent with a role for DPM neurons in directly enhancing potentiation. To test the sign of the connection, we first used functional imaging techniques to determine if DPM activation could stimulate postsynaptic MB neurons (Figure 4A). We expressed the mammalian ATP-gated P2X2 receptor (Lima and Miesenbock, 2005; Yao et al., 2012) in DPMs using NP2721-GAL4 or c316-GAL4, and activated these cells by applying ATP to dissected adult Drosophila brains. We first confirmed that bath-applied ATP was sufficient to activate the P2X2 receptors in DPMs by co-expressing genetically-encoded fluorescent sensors and using functional imaging to observe changes in fluorescence indicating a response. Using c316-GAL4 to drive UAS-GCaMP3.0 (Tian et al., 2009), UAS-Arclight (Cao et al., 2013), and UAS-Synapto-pHlorin (SpH) (Meisenbock et al., 1998), we found that P2X2-mediated stimulation effectively activated DPM neurons, evoking increases in intracellular calcium, membrane voltage, and vesicle fusion, respectively (Figure 4B). It should be noted that these responses were observed in the DPM projections to the MBs, not the DPM cell bodies, demonstrating that this technique successfully activates the DPMs and causes them to release neurotransmitter from their projections onto downstream targets in the MB neuropil. We also co-expressed P2X2 receptors and UAS-GCaMP3.0 in the DPMs using NP2721-GAL4 to confirm that this technique was effective with the weaker driver (Figure 4—figure supplement 1A).10.7554/eLife.03868.013Figure 4.DPM activation has no excitatory effect on the MBs.

Bottom Line: Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition.Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy.These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Volen Center for Complex Systems, National Center for Behavioral Genomics, Brandeis University, Waltham, United States.

ABSTRACT
Sleep promotes memory consolidation in humans and many other species, but the physiological and anatomical relationships between sleep and memory remain unclear. Here, we show the dorsal paired medial (DPM) neurons, which are required for memory consolidation in Drosophila, are sleep-promoting inhibitory neurons. DPMs increase sleep via release of GABA onto wake-promoting mushroom body (MB) α'/β' neurons. Functional imaging demonstrates that DPM activation evokes robust increases in chloride in MB neurons, but is unable to cause detectable increases in calcium or cAMP. Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition. Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy. These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.

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