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Phase coupling of a circadian neuropeptide with rest/activity rhythms detected using a membrane-tethered spider toxin.

Wu Y, Cao G, Pavlicek B, Luo X, Nitabach MN - PLoS Biol. (2008)

Bottom Line: These in vitro and in vivo electrophysiological effects of membrane-tethered delta-ACTX-Hv1a are consistent with the effects of soluble delta-ACTX-Hv1a purified from venom on Na(+) channel physiological and biophysical properties in cockroach neurons.Membrane-tethered delta-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops.As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA.

ABSTRACT
Drosophila clock neurons are self-sustaining cellular oscillators that rely on negative transcriptional feedback to keep circadian time. Proper regulation of organismal rhythms of physiology and behavior requires coordination of the oscillations of individual clock neurons within the circadian control network. Over the last decade, it has become clear that a key mechanism for intercellular communication in the circadian network is signaling between a subset of clock neurons that secrete the neuropeptide pigment dispersing factor (PDF) and clock neurons that possess its G protein-coupled receptor (PDFR). Furthermore, the specific hypothesis has been proposed that PDF-secreting clock neurons entrain the phase of organismal rhythms, and the cellular oscillations of other clock neurons, via the temporal patterning of secreted PDF signals. In order to test this hypothesis, we have devised a novel technique for altering the phase relationship between circadian transcriptional feedback oscillation and PDF secretion by using an ion channel-directed spider toxin to modify voltage-gated Na(+) channel inactivation in vivo. This technique relies on the previously reported "tethered-toxin" technology for cell-autonomous modulation of ionic conductances via heterologous expression of subtype-specific peptide ion channel toxins as chimeric fusion proteins tethered to the plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. We demonstrate for the first time, to our knowledge, the utility of the tethered-toxin technology in a transgenic animal, validating four different tethered spider toxin ion channel modifiers for use in Drosophila. Focusing on one of these toxins, we show that GPI-tethered Australian funnel-web spider toxin delta-ACTX-Hv1a inhibits Drosophila para voltage-gated Na(+) channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered delta-ACTX-Hv1a in vivo in the PDF-secreting subset of clock neurons induces rhythmic action potential bursts and depolarized plateau potentials. These in vitro and in vivo electrophysiological effects of membrane-tethered delta-ACTX-Hv1a are consistent with the effects of soluble delta-ACTX-Hv1a purified from venom on Na(+) channel physiological and biophysical properties in cockroach neurons. Membrane-tethered delta-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops. As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops. These results (1) validate the tethered-toxin technology for cell-autonomous modulation of ion channel biophysical properties in vivo in transgenic Drosophila, (2) demonstrate that the kinetics of para Na(+) channel inactivation is a key parameter for determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and (3) provide experimental support for the hypothesis that PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.

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Immunofluorescence Detection of Membrane-Tethered δ-ACTX-Hv1a in PDF-Secreting LNV Clock NeuronsAdult brains of pdf>δ-ACTX-Hv1a flies possessing the indicated number of UAS transgenes and the pdf-GAL4 transgene, or flies only possessing the indicated number of UAS-δ-ACTX-Hv1a transgenes, were processed for immunofluorescence with anti-Myc and anti-PDF antibodies to visualize both Myc epitope-tagged membrane-tethered δ-ACTX-Hv1a and PDF neuropeptide.(A) pdf>δ-ACTX-Hv1a (6×UAS) flies exhibit red anti-Myc immunofluorescence in the cell bodies of small LNVs (sLNVs) and large LNVs (lLNVs), sLNV dorsomedial terminals, and lLNV projections to the opposite optic lobe (not shown in this figure). Anti-Myc immunofluorescence colocalizes with green anti-PDF in the cell bodies of PDF neurons. Red anti-Myc immunofluorescence exhibits punctate staining throughout the sLNV terminals.(B) Bar graph demonstrates the dose-dependent expression of δ-ACTX-Hv1a in the lLNVs and sLNVs (p < 0.001; ANOVA Tukey-Kramer multiple comparisons). The intensity level (mean ± SEM) of anti-Myc labeling in the lLNVs and sLNVs is normalized to the intensity level in flies with six copies of UAS-δ-ACTX-Hv1a expressed with pdf-GAL4 driver. Bar graph for sLNV terminals represents percentage of brain hemispheres exhibiting anti-Myc staining in the sLNV terminals.
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pbio-0060273-g004: Immunofluorescence Detection of Membrane-Tethered δ-ACTX-Hv1a in PDF-Secreting LNV Clock NeuronsAdult brains of pdf>δ-ACTX-Hv1a flies possessing the indicated number of UAS transgenes and the pdf-GAL4 transgene, or flies only possessing the indicated number of UAS-δ-ACTX-Hv1a transgenes, were processed for immunofluorescence with anti-Myc and anti-PDF antibodies to visualize both Myc epitope-tagged membrane-tethered δ-ACTX-Hv1a and PDF neuropeptide.(A) pdf>δ-ACTX-Hv1a (6×UAS) flies exhibit red anti-Myc immunofluorescence in the cell bodies of small LNVs (sLNVs) and large LNVs (lLNVs), sLNV dorsomedial terminals, and lLNV projections to the opposite optic lobe (not shown in this figure). Anti-Myc immunofluorescence colocalizes with green anti-PDF in the cell bodies of PDF neurons. Red anti-Myc immunofluorescence exhibits punctate staining throughout the sLNV terminals.(B) Bar graph demonstrates the dose-dependent expression of δ-ACTX-Hv1a in the lLNVs and sLNVs (p < 0.001; ANOVA Tukey-Kramer multiple comparisons). The intensity level (mean ± SEM) of anti-Myc labeling in the lLNVs and sLNVs is normalized to the intensity level in flies with six copies of UAS-δ-ACTX-Hv1a expressed with pdf-GAL4 driver. Bar graph for sLNV terminals represents percentage of brain hemispheres exhibiting anti-Myc staining in the sLNV terminals.

Mentions: To confirm membrane targeting of membrane-tethered δ-ACTX-Hv1a in vivo, we examined the expression of Myc-tagged δ-ACTX-Hv1a in PDF-secreting clock neurons. The ten-amino acid Myc epitope tag is located in the middle of the glycine-asparagine repeat hydrophilic linker domain. Brains of flies with two, four, or six copies of UAS-δ-ACTX-Hv1a and pdf-GAL4 were double immunostained with anti-Myc and anti-PDF fluorescence, with parental 4×UAS-δ-ACTX-Hv1a flies as negative control for anti-Myc staining. As shown in Figure 4, control UAS-δ-ACTX-Hv1a flies show no anti-Myc immunofluorescence in the LNV cell bodies or terminals. Anti-PDF labels the cell bodies of PDF neurons (LNVs) and their processes, including dorsomedial terminals of sLNVs (Figure 4A) and projection of large LNVs (lLNVs) to the contralateral optic lobe through posterior optic tract (unpublished data). Flies expressing δ-ACTX-Hv1a in LNVs exhibit red anti-Myc immunofluorescence colocalized with anti-PDF green fluorescence in the cell bodies of LNVs. δ-ACTX-Hv1a expression dose-dependently increases anti-Myc immunofluorescence detected in the cell bodies of both lLNVs and sLNVs, as well as sLNV dorsomedial terminals (Figure 4). Red anti-Myc immunofluorescence is barely detectable in cell bodies of lLNV in pdf>2×UAS-δ-ACTX-Hv1a flies, and not at all in the sLNVs. Introduction of four or six copies of UAS-δ-ACTX-Hv1a transgene significantly increases anti-Myc immunofluorescence detected in the cell bodies of both lLNVs and sLNVs (p < 0.001, Figure 4). Anti-Myc immunofluorescence in sLNV dorsomedial terminals is not seen in brain hemispheres of pdf>2×UAS-δ-ACTX-Hv1a or pdf>4×UAS-δ-ACTX-Hv1a 4 flies. In pdf>6×UAS-δ-ACTX-Hv1a flies, 11 out of 16 hemispheres (68.8%) exhibit detectable anti-Myc immunofluorescence in the sLNV dorsomedial terminals (Figure 4B). Projection of lLNVs to the opposite optic lobes through the posterior optic tract is also visualized with anti-Myc immunofluorescence in these brains (unpublished data). These results establish that membrane-tethered δ-ACTX-Hv1a is expressed in LNV clock neurons and is transported throughout their neuronal arbors. Membrane-tethered ω-ACTX-Hv1c, a Ca2+ channel blocker, expression in LNVs is shown in Figure S1. High-resolution confocal images of the dorsomedial terminals of sLNVs expressing membrane-tethered ω-ACTX-Hv1c demonstrate green anti-Myc fluorescence signal surrounding “puncta” of red anti-PDF signal. This is consistent with targeting of ω-ACTX-Hv1c to the plasma membrane of the terminals that encloses regions of concentration of PDF-containing dense-core vesicles. These results indicate that membrane-tethered spider toxins are expressed by LNV clock neurons and targeted to the plasma membrane where they can interact with their target ion channels.


Phase coupling of a circadian neuropeptide with rest/activity rhythms detected using a membrane-tethered spider toxin.

Wu Y, Cao G, Pavlicek B, Luo X, Nitabach MN - PLoS Biol. (2008)

Immunofluorescence Detection of Membrane-Tethered δ-ACTX-Hv1a in PDF-Secreting LNV Clock NeuronsAdult brains of pdf>δ-ACTX-Hv1a flies possessing the indicated number of UAS transgenes and the pdf-GAL4 transgene, or flies only possessing the indicated number of UAS-δ-ACTX-Hv1a transgenes, were processed for immunofluorescence with anti-Myc and anti-PDF antibodies to visualize both Myc epitope-tagged membrane-tethered δ-ACTX-Hv1a and PDF neuropeptide.(A) pdf>δ-ACTX-Hv1a (6×UAS) flies exhibit red anti-Myc immunofluorescence in the cell bodies of small LNVs (sLNVs) and large LNVs (lLNVs), sLNV dorsomedial terminals, and lLNV projections to the opposite optic lobe (not shown in this figure). Anti-Myc immunofluorescence colocalizes with green anti-PDF in the cell bodies of PDF neurons. Red anti-Myc immunofluorescence exhibits punctate staining throughout the sLNV terminals.(B) Bar graph demonstrates the dose-dependent expression of δ-ACTX-Hv1a in the lLNVs and sLNVs (p < 0.001; ANOVA Tukey-Kramer multiple comparisons). The intensity level (mean ± SEM) of anti-Myc labeling in the lLNVs and sLNVs is normalized to the intensity level in flies with six copies of UAS-δ-ACTX-Hv1a expressed with pdf-GAL4 driver. Bar graph for sLNV terminals represents percentage of brain hemispheres exhibiting anti-Myc staining in the sLNV terminals.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2577701&req=5

pbio-0060273-g004: Immunofluorescence Detection of Membrane-Tethered δ-ACTX-Hv1a in PDF-Secreting LNV Clock NeuronsAdult brains of pdf>δ-ACTX-Hv1a flies possessing the indicated number of UAS transgenes and the pdf-GAL4 transgene, or flies only possessing the indicated number of UAS-δ-ACTX-Hv1a transgenes, were processed for immunofluorescence with anti-Myc and anti-PDF antibodies to visualize both Myc epitope-tagged membrane-tethered δ-ACTX-Hv1a and PDF neuropeptide.(A) pdf>δ-ACTX-Hv1a (6×UAS) flies exhibit red anti-Myc immunofluorescence in the cell bodies of small LNVs (sLNVs) and large LNVs (lLNVs), sLNV dorsomedial terminals, and lLNV projections to the opposite optic lobe (not shown in this figure). Anti-Myc immunofluorescence colocalizes with green anti-PDF in the cell bodies of PDF neurons. Red anti-Myc immunofluorescence exhibits punctate staining throughout the sLNV terminals.(B) Bar graph demonstrates the dose-dependent expression of δ-ACTX-Hv1a in the lLNVs and sLNVs (p < 0.001; ANOVA Tukey-Kramer multiple comparisons). The intensity level (mean ± SEM) of anti-Myc labeling in the lLNVs and sLNVs is normalized to the intensity level in flies with six copies of UAS-δ-ACTX-Hv1a expressed with pdf-GAL4 driver. Bar graph for sLNV terminals represents percentage of brain hemispheres exhibiting anti-Myc staining in the sLNV terminals.
Mentions: To confirm membrane targeting of membrane-tethered δ-ACTX-Hv1a in vivo, we examined the expression of Myc-tagged δ-ACTX-Hv1a in PDF-secreting clock neurons. The ten-amino acid Myc epitope tag is located in the middle of the glycine-asparagine repeat hydrophilic linker domain. Brains of flies with two, four, or six copies of UAS-δ-ACTX-Hv1a and pdf-GAL4 were double immunostained with anti-Myc and anti-PDF fluorescence, with parental 4×UAS-δ-ACTX-Hv1a flies as negative control for anti-Myc staining. As shown in Figure 4, control UAS-δ-ACTX-Hv1a flies show no anti-Myc immunofluorescence in the LNV cell bodies or terminals. Anti-PDF labels the cell bodies of PDF neurons (LNVs) and their processes, including dorsomedial terminals of sLNVs (Figure 4A) and projection of large LNVs (lLNVs) to the contralateral optic lobe through posterior optic tract (unpublished data). Flies expressing δ-ACTX-Hv1a in LNVs exhibit red anti-Myc immunofluorescence colocalized with anti-PDF green fluorescence in the cell bodies of LNVs. δ-ACTX-Hv1a expression dose-dependently increases anti-Myc immunofluorescence detected in the cell bodies of both lLNVs and sLNVs, as well as sLNV dorsomedial terminals (Figure 4). Red anti-Myc immunofluorescence is barely detectable in cell bodies of lLNV in pdf>2×UAS-δ-ACTX-Hv1a flies, and not at all in the sLNVs. Introduction of four or six copies of UAS-δ-ACTX-Hv1a transgene significantly increases anti-Myc immunofluorescence detected in the cell bodies of both lLNVs and sLNVs (p < 0.001, Figure 4). Anti-Myc immunofluorescence in sLNV dorsomedial terminals is not seen in brain hemispheres of pdf>2×UAS-δ-ACTX-Hv1a or pdf>4×UAS-δ-ACTX-Hv1a 4 flies. In pdf>6×UAS-δ-ACTX-Hv1a flies, 11 out of 16 hemispheres (68.8%) exhibit detectable anti-Myc immunofluorescence in the sLNV dorsomedial terminals (Figure 4B). Projection of lLNVs to the opposite optic lobes through the posterior optic tract is also visualized with anti-Myc immunofluorescence in these brains (unpublished data). These results establish that membrane-tethered δ-ACTX-Hv1a is expressed in LNV clock neurons and is transported throughout their neuronal arbors. Membrane-tethered ω-ACTX-Hv1c, a Ca2+ channel blocker, expression in LNVs is shown in Figure S1. High-resolution confocal images of the dorsomedial terminals of sLNVs expressing membrane-tethered ω-ACTX-Hv1c demonstrate green anti-Myc fluorescence signal surrounding “puncta” of red anti-PDF signal. This is consistent with targeting of ω-ACTX-Hv1c to the plasma membrane of the terminals that encloses regions of concentration of PDF-containing dense-core vesicles. These results indicate that membrane-tethered spider toxins are expressed by LNV clock neurons and targeted to the plasma membrane where they can interact with their target ion channels.

Bottom Line: These in vitro and in vivo electrophysiological effects of membrane-tethered delta-ACTX-Hv1a are consistent with the effects of soluble delta-ACTX-Hv1a purified from venom on Na(+) channel physiological and biophysical properties in cockroach neurons.Membrane-tethered delta-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops.As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA.

ABSTRACT
Drosophila clock neurons are self-sustaining cellular oscillators that rely on negative transcriptional feedback to keep circadian time. Proper regulation of organismal rhythms of physiology and behavior requires coordination of the oscillations of individual clock neurons within the circadian control network. Over the last decade, it has become clear that a key mechanism for intercellular communication in the circadian network is signaling between a subset of clock neurons that secrete the neuropeptide pigment dispersing factor (PDF) and clock neurons that possess its G protein-coupled receptor (PDFR). Furthermore, the specific hypothesis has been proposed that PDF-secreting clock neurons entrain the phase of organismal rhythms, and the cellular oscillations of other clock neurons, via the temporal patterning of secreted PDF signals. In order to test this hypothesis, we have devised a novel technique for altering the phase relationship between circadian transcriptional feedback oscillation and PDF secretion by using an ion channel-directed spider toxin to modify voltage-gated Na(+) channel inactivation in vivo. This technique relies on the previously reported "tethered-toxin" technology for cell-autonomous modulation of ionic conductances via heterologous expression of subtype-specific peptide ion channel toxins as chimeric fusion proteins tethered to the plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. We demonstrate for the first time, to our knowledge, the utility of the tethered-toxin technology in a transgenic animal, validating four different tethered spider toxin ion channel modifiers for use in Drosophila. Focusing on one of these toxins, we show that GPI-tethered Australian funnel-web spider toxin delta-ACTX-Hv1a inhibits Drosophila para voltage-gated Na(+) channel inactivation when coexpressed in Xenopus oocytes. Transgenic expression of membrane-tethered delta-ACTX-Hv1a in vivo in the PDF-secreting subset of clock neurons induces rhythmic action potential bursts and depolarized plateau potentials. These in vitro and in vivo electrophysiological effects of membrane-tethered delta-ACTX-Hv1a are consistent with the effects of soluble delta-ACTX-Hv1a purified from venom on Na(+) channel physiological and biophysical properties in cockroach neurons. Membrane-tethered delta-ACTX-Hv1a expression in the PDF-secreting subset of clock neurons induces an approximately 4-h phase advance of the rhythm of PDF accumulation in their terminals relative to both the phase of the day:night cycle and the phase of the circadian transcriptional feedback loops. As a consequence, the morning anticipatory peak of locomotor activity preceding dawn, which has been shown to be driven by the clocks of the PDF-secreting subset of clock neurons, phase advances coordinately with the phase of the PDF rhythm of the PDF-secreting clock neurons, rather than maintaining its phase relationship with the day:night cycle and circadian transcriptional feedback loops. These results (1) validate the tethered-toxin technology for cell-autonomous modulation of ion channel biophysical properties in vivo in transgenic Drosophila, (2) demonstrate that the kinetics of para Na(+) channel inactivation is a key parameter for determining the phase relationship between circadian transcriptional feedback oscillation and PDF secretion, and (3) provide experimental support for the hypothesis that PDF-secreting clock neurons entrain the phase of organismal rhythms via the temporal patterning of secreted PDF signals.

Show MeSH
Related in: MedlinePlus