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Function of the Shaw potassium channel within the Drosophila circadian clock.

Hodge JJ, Stanewsky R - PLoS ONE (2008)

Bottom Line: However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior.In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, Bristol University, Bristol, United Kingdom. james.hodge@bristol.ac.uk

ABSTRACT

Background: In addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K(+)) channel known to regulate RMP in Drosophila central neurons.

Methodology/principal findings: We show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN(1), DN(2) and DN(3)), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.

Conclusions/significance: Based on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.

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Changing Shaw levels in all clock neurons effects cyclic accumulation of PDF in LNv terminals.(A) Confocal image of the dorsal projections of the LNv neurons revealed by anti-PDF antibodies. The panels show representative samples of adult brains containing clock neurons with normal levels of Shaw (UAS-Shaw/CyO), elevated levels of Shaw (tim-GAL4/UAS-Shaw), a dominant-negative form of Shaw (tim-GAL4/UAS-ShawTR), or reduced levels of Shaw (tim-GAL4/UAS-Shaw-RNAi) that were fixed at the two time points indicated (ZT9 and ZT21). (B) Quantification of average staining intensity of the dorsal LNv projections from a single experiment (n = 7 to 10 hemispheres). Error bars represent SEM. In control brains PDF accumulation is higher during the day compared to the night. This circadian difference in PDF intensity is not seen when Shaw levels are manipulated in the clock neurons using tim-GAL4. In addition PDF tends to accumulate to higher levels compared to wild-type when Shaw levels are increased, while reduction or dominant-negative forms of Shaw tend to have the opposite effect.
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pone-0002274-g006: Changing Shaw levels in all clock neurons effects cyclic accumulation of PDF in LNv terminals.(A) Confocal image of the dorsal projections of the LNv neurons revealed by anti-PDF antibodies. The panels show representative samples of adult brains containing clock neurons with normal levels of Shaw (UAS-Shaw/CyO), elevated levels of Shaw (tim-GAL4/UAS-Shaw), a dominant-negative form of Shaw (tim-GAL4/UAS-ShawTR), or reduced levels of Shaw (tim-GAL4/UAS-Shaw-RNAi) that were fixed at the two time points indicated (ZT9 and ZT21). (B) Quantification of average staining intensity of the dorsal LNv projections from a single experiment (n = 7 to 10 hemispheres). Error bars represent SEM. In control brains PDF accumulation is higher during the day compared to the night. This circadian difference in PDF intensity is not seen when Shaw levels are manipulated in the clock neurons using tim-GAL4. In addition PDF tends to accumulate to higher levels compared to wild-type when Shaw levels are increased, while reduction or dominant-negative forms of Shaw tend to have the opposite effect.

Mentions: A mechanism by which Shaw K+ channels could influence the output of the clock independent of any effect on the molecular rhythm is by affecting PDF release from clock neurons. PDF release from Drosophila clock neurons is circadianly regulated and controls output behavior [26]. In order to determine if changing the level of Shaw in clock neurons affects rhythmic accumulation of PDF in the dorsal projections of s-LNv neurons, we quantified PDF levels in LNv terminals in the dorsal brain during a LD cycle (Figure 6). In wild-type animals there is more PDF in the LNv terminals during the day (ZT1 or ZT9) than at night (ZT13 or ZT21; p<0.00001) (Figure 6A–B; cf [26]). This circadian variation in PDF intensity is abolished when Shaw levels are manipulated in the clock neurons using tim-GAL4. Shaw over-expression in all clock neurons results in overall increased PDF levels compared to wild-type (ZT1 p<0.05, ZT9 and ZT21 p<0.00001), which are approximately the same at ZT1 and ZT21, and reaching peak and trough levels at ZT9 or ZT13, respectively (p<0.00001). Expression of dominant negative Shaw causes lower PDF levels at day and night compared to wild-type (ZT9 p<0.00001), but levels are somewhat higher at ZT21 and ZT13 compared to ZT9 (p<0.001 and p<0.05 respectively). Reducing Shaw in all clock neurons using Shaw RNAi also results in an overall reduction of PDF levels (ZT9 p<0.00001, ZT13 p<0.05) and peak levels are also reached during the night (p<0.0001). We cannot rule out that there might still be some PDF rhythm after Shaw over-expression, and possibly still residual, but improperly phased rhythms in flies with reduced Shaw.


Function of the Shaw potassium channel within the Drosophila circadian clock.

Hodge JJ, Stanewsky R - PLoS ONE (2008)

Changing Shaw levels in all clock neurons effects cyclic accumulation of PDF in LNv terminals.(A) Confocal image of the dorsal projections of the LNv neurons revealed by anti-PDF antibodies. The panels show representative samples of adult brains containing clock neurons with normal levels of Shaw (UAS-Shaw/CyO), elevated levels of Shaw (tim-GAL4/UAS-Shaw), a dominant-negative form of Shaw (tim-GAL4/UAS-ShawTR), or reduced levels of Shaw (tim-GAL4/UAS-Shaw-RNAi) that were fixed at the two time points indicated (ZT9 and ZT21). (B) Quantification of average staining intensity of the dorsal LNv projections from a single experiment (n = 7 to 10 hemispheres). Error bars represent SEM. In control brains PDF accumulation is higher during the day compared to the night. This circadian difference in PDF intensity is not seen when Shaw levels are manipulated in the clock neurons using tim-GAL4. In addition PDF tends to accumulate to higher levels compared to wild-type when Shaw levels are increased, while reduction or dominant-negative forms of Shaw tend to have the opposite effect.
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Related In: Results  -  Collection

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pone-0002274-g006: Changing Shaw levels in all clock neurons effects cyclic accumulation of PDF in LNv terminals.(A) Confocal image of the dorsal projections of the LNv neurons revealed by anti-PDF antibodies. The panels show representative samples of adult brains containing clock neurons with normal levels of Shaw (UAS-Shaw/CyO), elevated levels of Shaw (tim-GAL4/UAS-Shaw), a dominant-negative form of Shaw (tim-GAL4/UAS-ShawTR), or reduced levels of Shaw (tim-GAL4/UAS-Shaw-RNAi) that were fixed at the two time points indicated (ZT9 and ZT21). (B) Quantification of average staining intensity of the dorsal LNv projections from a single experiment (n = 7 to 10 hemispheres). Error bars represent SEM. In control brains PDF accumulation is higher during the day compared to the night. This circadian difference in PDF intensity is not seen when Shaw levels are manipulated in the clock neurons using tim-GAL4. In addition PDF tends to accumulate to higher levels compared to wild-type when Shaw levels are increased, while reduction or dominant-negative forms of Shaw tend to have the opposite effect.
Mentions: A mechanism by which Shaw K+ channels could influence the output of the clock independent of any effect on the molecular rhythm is by affecting PDF release from clock neurons. PDF release from Drosophila clock neurons is circadianly regulated and controls output behavior [26]. In order to determine if changing the level of Shaw in clock neurons affects rhythmic accumulation of PDF in the dorsal projections of s-LNv neurons, we quantified PDF levels in LNv terminals in the dorsal brain during a LD cycle (Figure 6). In wild-type animals there is more PDF in the LNv terminals during the day (ZT1 or ZT9) than at night (ZT13 or ZT21; p<0.00001) (Figure 6A–B; cf [26]). This circadian variation in PDF intensity is abolished when Shaw levels are manipulated in the clock neurons using tim-GAL4. Shaw over-expression in all clock neurons results in overall increased PDF levels compared to wild-type (ZT1 p<0.05, ZT9 and ZT21 p<0.00001), which are approximately the same at ZT1 and ZT21, and reaching peak and trough levels at ZT9 or ZT13, respectively (p<0.00001). Expression of dominant negative Shaw causes lower PDF levels at day and night compared to wild-type (ZT9 p<0.00001), but levels are somewhat higher at ZT21 and ZT13 compared to ZT9 (p<0.001 and p<0.05 respectively). Reducing Shaw in all clock neurons using Shaw RNAi also results in an overall reduction of PDF levels (ZT9 p<0.00001, ZT13 p<0.05) and peak levels are also reached during the night (p<0.0001). We cannot rule out that there might still be some PDF rhythm after Shaw over-expression, and possibly still residual, but improperly phased rhythms in flies with reduced Shaw.

Bottom Line: However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior.In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, Bristol University, Bristol, United Kingdom. james.hodge@bristol.ac.uk

ABSTRACT

Background: In addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K(+)) channel known to regulate RMP in Drosophila central neurons.

Methodology/principal findings: We show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN(1), DN(2) and DN(3)), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.

Conclusions/significance: Based on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.

Show MeSH
Related in: MedlinePlus