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Signal analysis of behavioral and molecular cycles.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Bottom Line: To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.We demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila.Using data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Brandeis University and NSF Center for Biological Timing, Waltham, MA 02454 USA. jlev@brandeis.edu

ABSTRACT

Background: Circadian clocks are biological oscillators that regulate molecular, physiological, and behavioral rhythms in a wide variety of organisms. While behavioral rhythms are typically monitored over many cycles, a similar approach to molecular rhythms was not possible until recently; the advent of real-time analysis using transgenic reporters now permits the observations of molecular rhythms over many cycles as well. This development suggests that new details about the relationship between molecular and behavioral rhythms may be revealed. Even so, behavioral and molecular rhythmicity have been analyzed using different methods, making such comparisons difficult to achieve. To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.

Results: We demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila. For both types of data, we apply digital filters to extract and clarify details of interest; we employ methods of autocorrelation and spectral analysis to assess rhythmicity and estimate the period; we evaluate phase shifts using crosscorrelation; and we use circular statistics to extract information about phase.

Conclusion: Using data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms. These methods are shown to be versatile and will also be adaptable to further experiments, owing in part to the non-proprietary nature of the code we have developed.

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Rhythms of Drosophila melanogaster. The fruitfly generates behavioral, molecular, and physiological rhythms on several different time scales. Examples (a) through (d) depict circadian rhythms, while (e) and (f) involve ultradian (high-frequency) cycles. The eclosion rhythm, plotted in (a) as numbers of emerging flies over time, is a population rhythm associated with metamorphosis from the pupal to the adult stage. In constant darkness (DD) emergence of adults from the pupal stage typically occurs in the early part of the subjective day, corresponding to literal daytime in light-dark (LD) cycles (alternating white and shaded blocks, left side of plot). (b) Adult behavioral rhythms are usually assayed by monitoring daily locomotor activity. The behavioral record shown is that of an adult wild-type male. The temporal distribution of activity was measured by the number of times he tripped an optical switch with the counts typically collected every half hour for several days. The photic conditions are depicted as in (a). (c) Activity of a firefly luciferase transgene driven by the timeless promoter. A transgenic male ingested luciferin substrate; and a rhythmic signal of bioluminescense was registered each hour, leading in this case to plotting of bioluminescent counts over a six-day timespan. (d) An isolated wing pair, dissected from the same type of transgenic fly as in (c), also displayed rhythmicity when bathed in a luciferin-containing medium. In this example the data are normalized as described in the text. (e) A one-second bout of male courtship song, the beginning of which (between 0 and 0.2 seconds) consisted of "sine" singing (generation of humming sounds by male wing vibrations). The sine-song episode proceeded into a train of tone pulses, which are produced at a rate of ca. 30 per second (by D. melanogaster males) such that the interpulse interval (I PI) is ca. 35 msec. An IPI rhythm is defined by systematic increases then decreases, etc., in the rates of pulse production; the duration of one such cycle is ca. one minute (in songs of this species). The ordinate for this song-bout plot is in arbitrary units, because this record reflects changes in voltage that have more to do with the monitoring equipment than the changes in pressure corresponding to varying sound levels per se. (f) A pupal cardiogram. The rhythmic heartbeat moves blood (hemolymph) throughout the open circulatory system of the animal. The motion of the heart muscle is plotted in arbitary units because the relative changes in voltage reflect changes in illumination (with respect to a non-invasive optical recording technique) produced by the muscle as it moves.
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Figure 1: Rhythms of Drosophila melanogaster. The fruitfly generates behavioral, molecular, and physiological rhythms on several different time scales. Examples (a) through (d) depict circadian rhythms, while (e) and (f) involve ultradian (high-frequency) cycles. The eclosion rhythm, plotted in (a) as numbers of emerging flies over time, is a population rhythm associated with metamorphosis from the pupal to the adult stage. In constant darkness (DD) emergence of adults from the pupal stage typically occurs in the early part of the subjective day, corresponding to literal daytime in light-dark (LD) cycles (alternating white and shaded blocks, left side of plot). (b) Adult behavioral rhythms are usually assayed by monitoring daily locomotor activity. The behavioral record shown is that of an adult wild-type male. The temporal distribution of activity was measured by the number of times he tripped an optical switch with the counts typically collected every half hour for several days. The photic conditions are depicted as in (a). (c) Activity of a firefly luciferase transgene driven by the timeless promoter. A transgenic male ingested luciferin substrate; and a rhythmic signal of bioluminescense was registered each hour, leading in this case to plotting of bioluminescent counts over a six-day timespan. (d) An isolated wing pair, dissected from the same type of transgenic fly as in (c), also displayed rhythmicity when bathed in a luciferin-containing medium. In this example the data are normalized as described in the text. (e) A one-second bout of male courtship song, the beginning of which (between 0 and 0.2 seconds) consisted of "sine" singing (generation of humming sounds by male wing vibrations). The sine-song episode proceeded into a train of tone pulses, which are produced at a rate of ca. 30 per second (by D. melanogaster males) such that the interpulse interval (I PI) is ca. 35 msec. An IPI rhythm is defined by systematic increases then decreases, etc., in the rates of pulse production; the duration of one such cycle is ca. one minute (in songs of this species). The ordinate for this song-bout plot is in arbitrary units, because this record reflects changes in voltage that have more to do with the monitoring equipment than the changes in pressure corresponding to varying sound levels per se. (f) A pupal cardiogram. The rhythmic heartbeat moves blood (hemolymph) throughout the open circulatory system of the animal. The motion of the heart muscle is plotted in arbitary units because the relative changes in voltage reflect changes in illumination (with respect to a non-invasive optical recording technique) produced by the muscle as it moves.

Mentions: In any metazoan organism, the timing system is increasingly appreciated to be complex. For example, whereas rhythmicity of locomotor activity is governed by a pacemaker within a discrete set of neurons in the Drosophila brain [8,9], molecular and physiological studies have also established the presence of autonomous circadian clocks in isolated appendages and excretory structures [10-12]. Moreover, the molecular mechanisms underlying clock function in these tissues may not be identical [13]. Finally, many rhythmic phenotypes expressed in flies can occur on different time scales. Figure 1 portrays 6 examples to illustrate the different levels of rhythmicity commonly studied in D. melanogaster. Each of these rhythms has heretofore been analyzed using a separate analytical technique. The periodic pattern of eclosion (emergence of the adult at the end of metamorphosis) and the pattern of adult locomotion (Figure 1a,b) are classic examples of circadian rhythms, which are typically analyzed by application of periodogram functions [14-17]. Figure 1c and 1d display examples of daily molecular rhythms for a whole fly and a pair of dissected wings, as reported in real time by luciferase-encoding DNA fused to regulatory sequences of the period (per) clock gene; these gene-product fluctuations have been analyzed using functions other than periodograms [10,18]. Drosophila rhythms are also evident on other time scales besides circa-24 hours. Courtship song, for example, consists of a series of sinusoidal hums and trains of pulses that come from the male's wing vibrations. The intervals between these pulses (interpulse intervals, or IPIs) have been shown to vary rhythmically with a period near one minute in D. melanogaster[19,20]. The trace shown in Figure 1e is taken from a digitized recording of the male courtship song, and such acoustical data have been subjected to still further kinds of time-series analysis [20-22]. Finally, at the highest frequency among these examples of periodic biological fluctuations, the heartbeat of the fruitfly exhibits a rhythm depicted in Figure 1f. It is driven by a pacemaker oscillator with a frequency on the order of about 2 Hz [23,24]. Although heartbeat, courtship song, and locomotor-activity rhythms occur on different time scales, the mechanisms underlying these different rhythms could be related. For example, mutations of the per gene affect both circadian and courtship song rhythms [19,20], but not heartbeat [25].


Signal analysis of behavioral and molecular cycles.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Rhythms of Drosophila melanogaster. The fruitfly generates behavioral, molecular, and physiological rhythms on several different time scales. Examples (a) through (d) depict circadian rhythms, while (e) and (f) involve ultradian (high-frequency) cycles. The eclosion rhythm, plotted in (a) as numbers of emerging flies over time, is a population rhythm associated with metamorphosis from the pupal to the adult stage. In constant darkness (DD) emergence of adults from the pupal stage typically occurs in the early part of the subjective day, corresponding to literal daytime in light-dark (LD) cycles (alternating white and shaded blocks, left side of plot). (b) Adult behavioral rhythms are usually assayed by monitoring daily locomotor activity. The behavioral record shown is that of an adult wild-type male. The temporal distribution of activity was measured by the number of times he tripped an optical switch with the counts typically collected every half hour for several days. The photic conditions are depicted as in (a). (c) Activity of a firefly luciferase transgene driven by the timeless promoter. A transgenic male ingested luciferin substrate; and a rhythmic signal of bioluminescense was registered each hour, leading in this case to plotting of bioluminescent counts over a six-day timespan. (d) An isolated wing pair, dissected from the same type of transgenic fly as in (c), also displayed rhythmicity when bathed in a luciferin-containing medium. In this example the data are normalized as described in the text. (e) A one-second bout of male courtship song, the beginning of which (between 0 and 0.2 seconds) consisted of "sine" singing (generation of humming sounds by male wing vibrations). The sine-song episode proceeded into a train of tone pulses, which are produced at a rate of ca. 30 per second (by D. melanogaster males) such that the interpulse interval (I PI) is ca. 35 msec. An IPI rhythm is defined by systematic increases then decreases, etc., in the rates of pulse production; the duration of one such cycle is ca. one minute (in songs of this species). The ordinate for this song-bout plot is in arbitrary units, because this record reflects changes in voltage that have more to do with the monitoring equipment than the changes in pressure corresponding to varying sound levels per se. (f) A pupal cardiogram. The rhythmic heartbeat moves blood (hemolymph) throughout the open circulatory system of the animal. The motion of the heart muscle is plotted in arbitary units because the relative changes in voltage reflect changes in illumination (with respect to a non-invasive optical recording technique) produced by the muscle as it moves.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65508&req=5

Figure 1: Rhythms of Drosophila melanogaster. The fruitfly generates behavioral, molecular, and physiological rhythms on several different time scales. Examples (a) through (d) depict circadian rhythms, while (e) and (f) involve ultradian (high-frequency) cycles. The eclosion rhythm, plotted in (a) as numbers of emerging flies over time, is a population rhythm associated with metamorphosis from the pupal to the adult stage. In constant darkness (DD) emergence of adults from the pupal stage typically occurs in the early part of the subjective day, corresponding to literal daytime in light-dark (LD) cycles (alternating white and shaded blocks, left side of plot). (b) Adult behavioral rhythms are usually assayed by monitoring daily locomotor activity. The behavioral record shown is that of an adult wild-type male. The temporal distribution of activity was measured by the number of times he tripped an optical switch with the counts typically collected every half hour for several days. The photic conditions are depicted as in (a). (c) Activity of a firefly luciferase transgene driven by the timeless promoter. A transgenic male ingested luciferin substrate; and a rhythmic signal of bioluminescense was registered each hour, leading in this case to plotting of bioluminescent counts over a six-day timespan. (d) An isolated wing pair, dissected from the same type of transgenic fly as in (c), also displayed rhythmicity when bathed in a luciferin-containing medium. In this example the data are normalized as described in the text. (e) A one-second bout of male courtship song, the beginning of which (between 0 and 0.2 seconds) consisted of "sine" singing (generation of humming sounds by male wing vibrations). The sine-song episode proceeded into a train of tone pulses, which are produced at a rate of ca. 30 per second (by D. melanogaster males) such that the interpulse interval (I PI) is ca. 35 msec. An IPI rhythm is defined by systematic increases then decreases, etc., in the rates of pulse production; the duration of one such cycle is ca. one minute (in songs of this species). The ordinate for this song-bout plot is in arbitrary units, because this record reflects changes in voltage that have more to do with the monitoring equipment than the changes in pressure corresponding to varying sound levels per se. (f) A pupal cardiogram. The rhythmic heartbeat moves blood (hemolymph) throughout the open circulatory system of the animal. The motion of the heart muscle is plotted in arbitary units because the relative changes in voltage reflect changes in illumination (with respect to a non-invasive optical recording technique) produced by the muscle as it moves.
Mentions: In any metazoan organism, the timing system is increasingly appreciated to be complex. For example, whereas rhythmicity of locomotor activity is governed by a pacemaker within a discrete set of neurons in the Drosophila brain [8,9], molecular and physiological studies have also established the presence of autonomous circadian clocks in isolated appendages and excretory structures [10-12]. Moreover, the molecular mechanisms underlying clock function in these tissues may not be identical [13]. Finally, many rhythmic phenotypes expressed in flies can occur on different time scales. Figure 1 portrays 6 examples to illustrate the different levels of rhythmicity commonly studied in D. melanogaster. Each of these rhythms has heretofore been analyzed using a separate analytical technique. The periodic pattern of eclosion (emergence of the adult at the end of metamorphosis) and the pattern of adult locomotion (Figure 1a,b) are classic examples of circadian rhythms, which are typically analyzed by application of periodogram functions [14-17]. Figure 1c and 1d display examples of daily molecular rhythms for a whole fly and a pair of dissected wings, as reported in real time by luciferase-encoding DNA fused to regulatory sequences of the period (per) clock gene; these gene-product fluctuations have been analyzed using functions other than periodograms [10,18]. Drosophila rhythms are also evident on other time scales besides circa-24 hours. Courtship song, for example, consists of a series of sinusoidal hums and trains of pulses that come from the male's wing vibrations. The intervals between these pulses (interpulse intervals, or IPIs) have been shown to vary rhythmically with a period near one minute in D. melanogaster[19,20]. The trace shown in Figure 1e is taken from a digitized recording of the male courtship song, and such acoustical data have been subjected to still further kinds of time-series analysis [20-22]. Finally, at the highest frequency among these examples of periodic biological fluctuations, the heartbeat of the fruitfly exhibits a rhythm depicted in Figure 1f. It is driven by a pacemaker oscillator with a frequency on the order of about 2 Hz [23,24]. Although heartbeat, courtship song, and locomotor-activity rhythms occur on different time scales, the mechanisms underlying these different rhythms could be related. For example, mutations of the per gene affect both circadian and courtship song rhythms [19,20], but not heartbeat [25].

Bottom Line: To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.We demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila.Using data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Brandeis University and NSF Center for Biological Timing, Waltham, MA 02454 USA. jlev@brandeis.edu

ABSTRACT

Background: Circadian clocks are biological oscillators that regulate molecular, physiological, and behavioral rhythms in a wide variety of organisms. While behavioral rhythms are typically monitored over many cycles, a similar approach to molecular rhythms was not possible until recently; the advent of real-time analysis using transgenic reporters now permits the observations of molecular rhythms over many cycles as well. This development suggests that new details about the relationship between molecular and behavioral rhythms may be revealed. Even so, behavioral and molecular rhythmicity have been analyzed using different methods, making such comparisons difficult to achieve. To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.

Results: We demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila. For both types of data, we apply digital filters to extract and clarify details of interest; we employ methods of autocorrelation and spectral analysis to assess rhythmicity and estimate the period; we evaluate phase shifts using crosscorrelation; and we use circular statistics to extract information about phase.

Conclusion: Using data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms. These methods are shown to be versatile and will also be adaptable to further experiments, owing in part to the non-proprietary nature of the code we have developed.

Show MeSH
Related in: MedlinePlus