Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet m... more Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau to rhythmic behavior remain unclear. Here, we find that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, we observe a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss.
Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulner... more Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. Here, we used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep.
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics. .
bioRxiv (Cold Spring Harbor Laboratory), Mar 3, 2022
The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for sett... more The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for setting behavioral phase especially under short photoperiod conditions. Yet the circadian clock neurons and underlying mechanisms important for these effects are unknown. Here we demonstrate that PRL-1 function in PDF neurons is necessary and sufficient for timing morning and evening phase under short photoperiod (6:18) conditions. In standard (12:12) photoperiod, we find that PRL-1 rescue or blocking PRL-1 knockdown in PDF neurons restores wild-type morning phase. Expression of the TIM kinase shaggy (sgg) in PDF neurons suppresses in an additive manner PRL-1 period lengthening and delayed morning phase further underscoring the role of PDF neurons. In contrast to knockdown in all clock neurons, knockdown of PRL-1 selectively in PDF clock neurons does not alter the timing of morning or evening phase under standard photoperiod conditions, suggesting PRL-1 function in non-PDF neurons may also be important. In contrast to SGG effects, we find that phase delay of evening onset due to expression of a dominant negative version of the TIM kinase CK2 (CK2alpha Tik ) requires PRL-1. Given that TIM is a substrate of both the PRL-1 phosphatase and CK2 kinase, we hypothesize that PRL-1 dephosphorylation at specific sites may be necessary for subsequent CK2 phosphorylation to time behavior.
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that c... more In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects. During the second round of review of this article, it came to light that the first author, under a pseudonym, had engaged in activities which do not align with PLOS' values. Please note that PLOS strongly condemns all discriminatory behaviors, attitudes, and actions.
bioRxiv (Cold Spring Harbor Laboratory), Feb 2, 2022
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics. .
Introduction: Insomnia is a common sleep disorder affecting approximately 10-30% of the populatio... more Introduction: Insomnia is a common sleep disorder affecting approximately 10-30% of the population. Although numerous studies have investigated the impact of insomnia on psychosocial functioning, there have been few published studies evaluating the neurophysiologic implications of insomnia. Examining resting cerebral blood flow (rCBF) in patients with Primary Insomnia (PI) is particularly essential as it will aid in elucidating the impact of insomnia on the neurophysiologic response to cognitive challenge. Methods: 9 patients who met DSM-IV-TR criteria for PI and additional research criteria (≥30mins to fall and/or stay asleep, and either <6 hours total sleep time [TST] or sleep efficiency [SE] <85% on ≥3 nights per week for ≥6 months, with daytime complaint) were matched to 9 Good Sleeper (GS) controls for gender, age (+/-8yrs), and education level (+/-2yrs) All subjects underwent 1-week baseline evaluation, followed by 2-nights polysomnography (PSG), 1-2 nights at-home sleep, and an imaging protocol consisting of fMRI tasks and assessment of rCBF using arterial spin labeling (ASL). Groups were compared on PSG and rCBF using independent samples t-tests. Bivariate correlations were performed for PSG on rCBF in regions found to significantly differ between groups. Results: Based on second night (baseline) PSG, PIs had significantly less TST (388min vs 439min; p=.033), greater wake after sleep onset time (WASO:65min vs 21min; p=.009), and lower SE (83% vs 93%; p=.012). ASL analyses determined that PIs exhibited lower rCBF in frontal, insular, occipital, and subcortical regions (p's<.01; uncorrected). Furthermore, increased WASO and poorer SE were found to be significantly associated with lower rCBF in the anterior insula p=.04; SE:r=.481, p=.04) and putamen (WASO:r=-.488, p=.04; SE:r=.504, p=.03); higher percent Stage 1 sleep was associated with lower rCBF in the left inferior frontal gyrus (r=-.474, p=.05). Conclusion: In this preliminary investigation, PIs exhibited a pattern of reduced rCBF in regions implicated in wide ranging functions including language and emotional processing and regulation of movement. Future research is needed to determine the short-and long-term implications of insomnia on neurophysiologic functioning.
Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. MARCH 2019 2. REPORT TYPE Final 3. DATES COVERED 1JUN2016 -30NOV2018 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic Sleep for Traumatic Brain Injury 5b. GRANT NUMBER W81XWH-16-1-0166 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Rav i Allada 5e. TASK NUMBER Bart van Alphen 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that c... more In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. S...
The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for sett... more The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for setting behavioral phase especially under short photoperiod conditions. Yet the circadian clock neurons and underlying mechanisms important for these effects are unknown. Here we demonstrate that PRL-1 function in PDF neurons is necessary and sufficient for timing morning and evening phase under short photoperiod (6:18) conditions. In standard (12:12) photoperiod, we find that PRL-1 rescue or blocking PRL-1 knockdown in PDF neurons restores wild-type morning phase. Expression of the TIM kinase shaggy (sgg) in PDF neurons suppresses in an additive manner PRL-1 period lengthening and delayed morning phase further underscoring the role of PDF neurons. In contrast to knockdown in all clock neurons, knockdown of PRL-1 selectively in PDF clock neurons does not alter the timing of morning or evening phase under standard photoperiod conditions, suggesting PRL-1 function in non-PDF neurons may also be important. In contrast to SGG effects, we find that phase delay of evening onset due to expression of a dominant negative version of the TIM kinase CK2 (CK2alpha Tik ) requires PRL-1. Given that TIM is a substrate of both the PRL-1 phosphatase and CK2 kinase, we hypothesize that PRL-1 dephosphorylation at specific sites may be necessary for subsequent CK2 phosphorylation to time behavior.
Proceedings of the National Academy of Sciences, 2021
Significance In animals, the control of daily sleep–wake rhythms is mediated by discrete circadia... more Significance In animals, the control of daily sleep–wake rhythms is mediated by discrete circadian clock neurons via their rhythmic activity–dependent release of neuropeptides and neurotransmitters. Here, we describe a gene, Tango10 , critical for daily behavioral rhythms. TANGO10 functions as an adaptor with its partner, the E3 ubiquitin ligase CULLIN 3, to regulate protein ubiquitination, neuronal excitability via voltage-gated potassium channels, and neuropeptide accumulation. These studies define a mechanism for control of neuronal excitability and daily rhythmicity.
The most enigmatic of the canonical properties of circadian clocks is temperature compensation wh... more The most enigmatic of the canonical properties of circadian clocks is temperature compensation where circadian period length is stable across a wide temperature range despite the temperature dependence of most biochemical reactions. While the core mechanisms of circadian clocks have been well described, the molecular mechanisms of temperature compensation are poorly understood especially in animals. A major gap is the lack of temperature compensation mutants that do not themselves unambiguously affect the temperature dependence of the encoded protein. Here we show that null alleles of two genes encoding components of a complex important for translation of the core clock component period in circadian pacemaker neurons robustly alter the temperature dependence of circadian behavioral period length. These changes are accompanied by parallel temperature dependent changes in oscillations of the PER protein and are consistent with the model that these translation factors mediate the tempe...
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics.
Anticipation Index. To calculate Anticipation Index values (Table S1), we determined the largest ... more Anticipation Index. To calculate Anticipation Index values (Table S1), we determined the largest two-hour increase in normalized average activity of each genotype over the entire12 hour light phase (Evening Anticipation Index) or 12 hour dark phase (Morning Anticipation Index). The time designation for anticipation (Table S1: ZT Time) refers to the end point of that two-hour period. All p values reported were calculated using Student’s two-tailed t-test with equal variance. Transgenic Drosophila strains. To generate UAS-na, two overlapping na cDNA fragments were amplified using Platinum Pfx Polymerase (Invitrogen) and the following gene-specific primers: forward primer 1, 5’-AGGTGGTCTCAATCTGCTCGG-3’; reverse primer 1, 5’-GTGACCAGACCGAGAAAGTTGTG-3’; forward primer 2, 5’-CTTTCGCAAGCAGAAGCCC-3’; reverse primer 2, 5’-TGGTTGCCCTTTTTGTTTCC-3 ’ (Integrated DNA Technologies). PCR products were cloned into TOPO-TA vectors (Invitrogen) and subcloned to generate a single larger na cDNA. An Eco...
SummaryHomeostatic and circadian processes collaborate to appropriately time and consolidate slee... more SummaryHomeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, we scheduled brief sleep deprivation at different times of day in Drosophila and find elevated morning rebound compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, we identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center.
Proboscis extension–mediated waste clearance during deep sleep in Drosophila reveals a conserved ... more Proboscis extension–mediated waste clearance during deep sleep in Drosophila reveals a conserved primordial function of sleep.
The timing of behavior under natural light-dark conditions is a function of circadian clocks and ... more The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.
F1000 - Post-publication peer review of the biomedical literature, 2016
In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pac... more In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brain-wide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca 2+ that were entrained by environmental cues and timed by molecular clocks. However, these rhythms were not synchronous, as each group exhibited its own phase of activation. Ca 2+ rhythms displayed by pacemaker groups that were associated with the morning or evening locomotor activities occurred ~4 hours before their respective behaviors. Loss of receptor for neuropeptide PDF promoted synchrony of Ca 2+ waves. Thus neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers. Circadian clocks help animals adapt their physiology and behavior to local time. The clocks require a highly-conserved set of genes and proteins (1) operating through molecular feedback loops to generate robust rhythms that produce a 24 hour timing signal (2). These clocks are expressed by pacemaker neurons which themselves are assembled into an interactive network (3). Through network encoding and cellular interactions, pacemaker neurons in the suprachiasmatic nucleus (SCN) of the mammalian brain coordinate many circadian rhythmic outputs (4-7). To study how molecular clocks couple to network encoding, and how network encoding relates to specific behavioral outputs, we conducted an in vivo brain-wide analysis of the circadian pacemaker network in Drosophila across an entire 24 hour day.
Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet m... more Disrupted circadian rhythms are a prominent feature of multiple neurodegenerative diseases. Yet mechanisms linking Tau to rhythmic behavior remain unclear. Here, we find that expression of a phosphomimetic human Tau mutant (TauE14) in Drosophila circadian pacemaker neurons disrupts free-running rhythmicity. While cell number and oscillations of the core clock protein PERIOD are unaffected in the small LNv (sLNv) neurons important for free running rhythms, we observe a near complete loss of the major LNv neuropeptide pigment dispersing factor (PDF) in the dorsal axonal projections of the sLNvs. This was accompanied by a ~50% reduction in the area of the dorsal terminals and a modest decrease in cell body PDF levels. Expression of wild-type Tau also reduced axonal PDF levels but to a lesser extent than TauE14. TauE14 also induces a complete loss of mitochondria from these sLNv projections. However, mitochondria were increased in sLNv cell bodies in TauE14 flies. These results suggest that TauE14 disrupts axonal transport of neuropeptides and mitochondria in circadian pacemaker neurons, providing a mechanism by which Tau can disrupt circadian behavior prior to cell loss.
Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulner... more Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. Here, we used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep.
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics. .
bioRxiv (Cold Spring Harbor Laboratory), Mar 3, 2022
The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for sett... more The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for setting behavioral phase especially under short photoperiod conditions. Yet the circadian clock neurons and underlying mechanisms important for these effects are unknown. Here we demonstrate that PRL-1 function in PDF neurons is necessary and sufficient for timing morning and evening phase under short photoperiod (6:18) conditions. In standard (12:12) photoperiod, we find that PRL-1 rescue or blocking PRL-1 knockdown in PDF neurons restores wild-type morning phase. Expression of the TIM kinase shaggy (sgg) in PDF neurons suppresses in an additive manner PRL-1 period lengthening and delayed morning phase further underscoring the role of PDF neurons. In contrast to knockdown in all clock neurons, knockdown of PRL-1 selectively in PDF clock neurons does not alter the timing of morning or evening phase under standard photoperiod conditions, suggesting PRL-1 function in non-PDF neurons may also be important. In contrast to SGG effects, we find that phase delay of evening onset due to expression of a dominant negative version of the TIM kinase CK2 (CK2alpha Tik ) requires PRL-1. Given that TIM is a substrate of both the PRL-1 phosphatase and CK2 kinase, we hypothesize that PRL-1 dephosphorylation at specific sites may be necessary for subsequent CK2 phosphorylation to time behavior.
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that c... more In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects. During the second round of review of this article, it came to light that the first author, under a pseudonym, had engaged in activities which do not align with PLOS' values. Please note that PLOS strongly condemns all discriminatory behaviors, attitudes, and actions.
bioRxiv (Cold Spring Harbor Laboratory), Feb 2, 2022
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics. .
Introduction: Insomnia is a common sleep disorder affecting approximately 10-30% of the populatio... more Introduction: Insomnia is a common sleep disorder affecting approximately 10-30% of the population. Although numerous studies have investigated the impact of insomnia on psychosocial functioning, there have been few published studies evaluating the neurophysiologic implications of insomnia. Examining resting cerebral blood flow (rCBF) in patients with Primary Insomnia (PI) is particularly essential as it will aid in elucidating the impact of insomnia on the neurophysiologic response to cognitive challenge. Methods: 9 patients who met DSM-IV-TR criteria for PI and additional research criteria (≥30mins to fall and/or stay asleep, and either <6 hours total sleep time [TST] or sleep efficiency [SE] <85% on ≥3 nights per week for ≥6 months, with daytime complaint) were matched to 9 Good Sleeper (GS) controls for gender, age (+/-8yrs), and education level (+/-2yrs) All subjects underwent 1-week baseline evaluation, followed by 2-nights polysomnography (PSG), 1-2 nights at-home sleep, and an imaging protocol consisting of fMRI tasks and assessment of rCBF using arterial spin labeling (ASL). Groups were compared on PSG and rCBF using independent samples t-tests. Bivariate correlations were performed for PSG on rCBF in regions found to significantly differ between groups. Results: Based on second night (baseline) PSG, PIs had significantly less TST (388min vs 439min; p=.033), greater wake after sleep onset time (WASO:65min vs 21min; p=.009), and lower SE (83% vs 93%; p=.012). ASL analyses determined that PIs exhibited lower rCBF in frontal, insular, occipital, and subcortical regions (p's<.01; uncorrected). Furthermore, increased WASO and poorer SE were found to be significantly associated with lower rCBF in the anterior insula p=.04; SE:r=.481, p=.04) and putamen (WASO:r=-.488, p=.04; SE:r=.504, p=.03); higher percent Stage 1 sleep was associated with lower rCBF in the left inferior frontal gyrus (r=-.474, p=.05). Conclusion: In this preliminary investigation, PIs exhibited a pattern of reduced rCBF in regions implicated in wide ranging functions including language and emotional processing and regulation of movement. Future research is needed to determine the short-and long-term implications of insomnia on neurophysiologic functioning.
Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. MARCH 2019 2. REPORT TYPE Final 3. DATES COVERED 1JUN2016 -30NOV2018 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic Sleep for Traumatic Brain Injury 5b. GRANT NUMBER W81XWH-16-1-0166 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Rav i Allada 5e. TASK NUMBER Bart van Alphen 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT
In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that c... more In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. S...
The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for sett... more The TIMELESS (TIM) phosphatase, Phosphatase of Regenerating Liver-1 (PRL-1), is required for setting behavioral phase especially under short photoperiod conditions. Yet the circadian clock neurons and underlying mechanisms important for these effects are unknown. Here we demonstrate that PRL-1 function in PDF neurons is necessary and sufficient for timing morning and evening phase under short photoperiod (6:18) conditions. In standard (12:12) photoperiod, we find that PRL-1 rescue or blocking PRL-1 knockdown in PDF neurons restores wild-type morning phase. Expression of the TIM kinase shaggy (sgg) in PDF neurons suppresses in an additive manner PRL-1 period lengthening and delayed morning phase further underscoring the role of PDF neurons. In contrast to knockdown in all clock neurons, knockdown of PRL-1 selectively in PDF clock neurons does not alter the timing of morning or evening phase under standard photoperiod conditions, suggesting PRL-1 function in non-PDF neurons may also be important. In contrast to SGG effects, we find that phase delay of evening onset due to expression of a dominant negative version of the TIM kinase CK2 (CK2alpha Tik ) requires PRL-1. Given that TIM is a substrate of both the PRL-1 phosphatase and CK2 kinase, we hypothesize that PRL-1 dephosphorylation at specific sites may be necessary for subsequent CK2 phosphorylation to time behavior.
Proceedings of the National Academy of Sciences, 2021
Significance In animals, the control of daily sleep–wake rhythms is mediated by discrete circadia... more Significance In animals, the control of daily sleep–wake rhythms is mediated by discrete circadian clock neurons via their rhythmic activity–dependent release of neuropeptides and neurotransmitters. Here, we describe a gene, Tango10 , critical for daily behavioral rhythms. TANGO10 functions as an adaptor with its partner, the E3 ubiquitin ligase CULLIN 3, to regulate protein ubiquitination, neuronal excitability via voltage-gated potassium channels, and neuropeptide accumulation. These studies define a mechanism for control of neuronal excitability and daily rhythmicity.
The most enigmatic of the canonical properties of circadian clocks is temperature compensation wh... more The most enigmatic of the canonical properties of circadian clocks is temperature compensation where circadian period length is stable across a wide temperature range despite the temperature dependence of most biochemical reactions. While the core mechanisms of circadian clocks have been well described, the molecular mechanisms of temperature compensation are poorly understood especially in animals. A major gap is the lack of temperature compensation mutants that do not themselves unambiguously affect the temperature dependence of the encoded protein. Here we show that null alleles of two genes encoding components of a complex important for translation of the core clock component period in circadian pacemaker neurons robustly alter the temperature dependence of circadian behavioral period length. These changes are accompanied by parallel temperature dependent changes in oscillations of the PER protein and are consistent with the model that these translation factors mediate the tempe...
Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular m... more Drosophila is a premier model for the study of circadian rhythms and sleep, revealing molecular mechanisms that are conserved with vertebrates, including humans. The Drosophila Activity Monitoring (DAM) system has been employed for automated, high throughput analyses, enabling measures of a wide range of measures of circadian and sleep, including period, rhythmicity, anticipation, sleep amount, bout length, and bout number. Yet there are no publicly available programs that capture the full breadth of these measures. Here we develop a new program SleepMat that analyzes a uniquely broader range of circadian and sleep parameters. The program is built on a Matlab platform, employs an easy-to-use graphic user interface, and is highly flexible to customize data inputs. This program will enable a user-friendly high throughput analysis of a broad range of sleep and circadian parameters that can be coupled to the power of Drosophila genetics.
Anticipation Index. To calculate Anticipation Index values (Table S1), we determined the largest ... more Anticipation Index. To calculate Anticipation Index values (Table S1), we determined the largest two-hour increase in normalized average activity of each genotype over the entire12 hour light phase (Evening Anticipation Index) or 12 hour dark phase (Morning Anticipation Index). The time designation for anticipation (Table S1: ZT Time) refers to the end point of that two-hour period. All p values reported were calculated using Student’s two-tailed t-test with equal variance. Transgenic Drosophila strains. To generate UAS-na, two overlapping na cDNA fragments were amplified using Platinum Pfx Polymerase (Invitrogen) and the following gene-specific primers: forward primer 1, 5’-AGGTGGTCTCAATCTGCTCGG-3’; reverse primer 1, 5’-GTGACCAGACCGAGAAAGTTGTG-3’; forward primer 2, 5’-CTTTCGCAAGCAGAAGCCC-3’; reverse primer 2, 5’-TGGTTGCCCTTTTTGTTTCC-3 ’ (Integrated DNA Technologies). PCR products were cloned into TOPO-TA vectors (Invitrogen) and subcloned to generate a single larger na cDNA. An Eco...
SummaryHomeostatic and circadian processes collaborate to appropriately time and consolidate slee... more SummaryHomeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, we scheduled brief sleep deprivation at different times of day in Drosophila and find elevated morning rebound compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, we identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center.
Proboscis extension–mediated waste clearance during deep sleep in Drosophila reveals a conserved ... more Proboscis extension–mediated waste clearance during deep sleep in Drosophila reveals a conserved primordial function of sleep.
The timing of behavior under natural light-dark conditions is a function of circadian clocks and ... more The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.
F1000 - Post-publication peer review of the biomedical literature, 2016
In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pac... more In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brain-wide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca 2+ that were entrained by environmental cues and timed by molecular clocks. However, these rhythms were not synchronous, as each group exhibited its own phase of activation. Ca 2+ rhythms displayed by pacemaker groups that were associated with the morning or evening locomotor activities occurred ~4 hours before their respective behaviors. Loss of receptor for neuropeptide PDF promoted synchrony of Ca 2+ waves. Thus neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers. Circadian clocks help animals adapt their physiology and behavior to local time. The clocks require a highly-conserved set of genes and proteins (1) operating through molecular feedback loops to generate robust rhythms that produce a 24 hour timing signal (2). These clocks are expressed by pacemaker neurons which themselves are assembled into an interactive network (3). Through network encoding and cellular interactions, pacemaker neurons in the suprachiasmatic nucleus (SCN) of the mammalian brain coordinate many circadian rhythmic outputs (4-7). To study how molecular clocks couple to network encoding, and how network encoding relates to specific behavioral outputs, we conducted an in vivo brain-wide analysis of the circadian pacemaker network in Drosophila across an entire 24 hour day.
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Papers by Ravi Allada