1,788 research outputs found
The Genetic and Neuronal Substrates of Melatonin Signaling in Zebrafish Sleep
Sleep is hypothesized to be regulated by two processes: a circadian drive, which communicates time of day to ensure that sleep is timed to the appropriate day/night phase, and a homeostatic drive, by which the propensity for sleep becomes stronger over the course of prolonged wakefulness. While studies suggest that adenosine and serotonin signaling in part mediate the homeostatic sleep drive, factors that act downstream of the circadian clock to promote sleep were unidentified until recently. Previous work in the Prober lab has shown that the nocturnal hormone melatonin acts downstream of the circadian rhythm to promote sleep in zebrafish. The downstream processes by which melatonin promotes sleep is poorly understood across all animal models. This is likely because melatonin research has been primarily conducted using nocturnal laboratory rodent models, in whom melatonin does not seem to play a role in sleep, and because of the widely held view that melatonin informs the circadian clock and does not promote sleep directly. In Chapter 1 of this thesis, I review some of the research conducted over the last 50 years that has informed our current understanding of melatonin and its role in sleep. In Chapter 2, I describe our efforts to use the zebrafish, in which melatonin is both potently sedating and essential for nightly sleep, to uncover some of the mechanisms by which melatonin might promote sleep. We found that melatonin acts through a particular melatonin receptor family called MT1, whereas melatonin receptors belonging to other families were dispensable for sleep. We show that MT1 receptors are expressed broadly throughout the zebrafish brain and are enriched in brain regions involved in sensory processing, particularly in those related to vision. We tested the hypothesis that melatonin promotes sleep, at least in part, by dampening visual responsiveness at night. We show that, separable from sleep, exogenous melatonin suppresses behavioral responses to light stimuli, and loss of endogenous melatonin results in day-like behavioral responses to light stimuli during the night. We are using whole brain imaging in live zebrafish to corroborate our behavioral results with neuronal GCaMP recordings. We hope that the findings presented here contribute to a greater understanding of melatonin’s role in sleep, which may help enhance its value as a natural therapeutic aid
Enabling neighbour-labelling: using synthetic biology to explore how cells influence their neighbours
Cell-cell interactions are central to development, but exploring how a change in any given cell relates to changes in the neighbour of that cell can be technically challenging. Here, we review recent developments in synthetic biology and image analysis that are helping overcome this problem. We highlight the opportunities presented by these advances and discuss opportunities and limitations in applying them to developmental model systems
Development of variable and robust brain wiring patterns in the fly visual system
Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear.
In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes.
In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain
Signaling Mechanisms Behind the Benefits of Sleep
Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende Würmer nach einer Verletzung häufiger. Insgesamt deutet dies darauf hin, dass AMP als Signalmoleküle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. Für diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlüsseln, durch den verschiedene Reize der angeborenen Immunität, das heißt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche Domänen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-Domäne als aktive Proteasedomäne von NAS-38 angesehen wird, erwartete ich eine Schlüsselrolle dieser Domäne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitätsinduziertem Schlaf? Da gezeigt wurde, dass AMP während des NAS-38 Schlafes und auch nach Verwundung hochreguliert sind, erwartete ich, dass AMP an der Signalisierung von Schlaf von der Epidermis zum Nervensystem beteiligt sind. In einem zweiten Schritt untersuchte ich die molekularen Mechanismen, die den Vorteilen von Schlaf für das Überleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: Verändert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf für viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeinträchtigt. Zweitens, ist Schlaf wichtig für die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? Während der Larvenentwicklung fällt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeinträchtigt. Methoden: Zur Analyse der Signalmechanismen, durch die sowohl NAS-38 als auch Verletzungen Schlaf induzieren, filmte ich das Schlafverhalten von C. elegans mittels Langzeit-Bildgebung in Agarose-Mikrokammern. So führte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 Domäne deletiert war. Darüber hinaus testete ich verschiedene Suppressoren für immunvermittelten Schlaf, der durch NAS 38 oder Verletzungen induziert war. Die Redundanz des Suppressionseffektes der verschiedenen Mitglieder der AMP-Familie auf immunvermittelten Schlaf testete ich, indem ich den Suppressionsphänotyp einer CRISPR/Cas9-editierten Multi-Knockout-Mutante analysierte, in der insgesamt 19 AMP deletiert waren. Um Effektoren zu identifizieren, die den AMP nachgeschaltet sind, induzierte ich Schlaf durch Überexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschäftigte ich mich mit der Frage, wie genau Schlaf das Überleben nach Verletzungen unterstützt. Ich verglich die Expression von literaturbekannten Reportern für verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell für chronisch-genetischen Schlafmangel. Darüber hinaus habe ich die Transkriptome zwischen jeweils adulten verwundeten und unverwundeten Wildtypen und schlaflosen Mutanten verglichen. Um die Struktur der Cuticula des Wildtyps und der schlaflosen Mutante zu vergleichen, analysierte ich außerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-Domäne verlängert. Dieser Prozess wird moderiert durch die TSP-1-Domäne. Weiterhin konnte ich zeigen, dass viele AMP redundant wirken um immunvermittelten Schlaf, verursacht durch NAS-38 oder Verletzungen, zu signalisieren. Ich konnte zeigen, dass das AMP NLP-29 über den Neuropeptidrezeptor NPR-12 wirkt. Dieser kann NLP-29-induzierten Schlaf vermitteln, wenn er in einem neuronalen Netzwerk exprimiert wird, welches nachweislich RIS aktiviert. Interessanterweise fand ich außerdem heraus, dass für NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verändert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verändert. Dies betraf unter anderem eine Gruppe oszillierender Gene, die Cuticula-assoziierte Proteine codieren, und deren Expression normalerweise ihren Höhepunkt gegen Ende des Lethargus erreicht. Da angenommen wird, dass der Zeitpunkt der Kollagenexpression entscheidend für eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsächlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die Strapazierfähigkeit der Cuticula gegenüber bestimmten Belastungen verringern. Daher könnte Schlaf erforderlich sein, Robustheit in Form einer strukturierten Cuticula zu fördern. Schlussfolgerungen: In diesem Dissertationsprojekt vollendete ich die Charakterisierung eines neuentdeckten Mechanismus in C. elegans, durch den Verwundungen Schlaf als Teil der Immunantwort aus der Peripherie zum Nervensystem signalisieren. Ich konnte zeigen, dass AMP gewebeübergreifend Signale von der Epidermis an ein neuronales Netz vermitteln, welches wiederum RIS aktiviert und dadurch Schlaf induziert. Da Komponenten dieses Signalweges konserviert sind, könnten AMP auch in anderen Tieren, einschließlich des Menschen, Schlaf zur Genesung fördern. Darüber hinaus habe ich die Grundlagen für die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes für Heilung und Überleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verändern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfältigere Rolle bei der zeitlichen Regulierung der Genexpression hin.:Summary I
Zusammenfassung IV
Contents VII
List of Figures XII
List of Tables XIV
Abbreviations XV
1. Introduction 1
1.1. Sleep is fascinating 1
1.1.1. The origin and basic features of sleep 1
1.1.2. Regulation of sleep in higher animals 3
1.1.2.1. Neuronal control of sleep 3
1.1.2.2. Molecular control of sleep 5
1.1.3. The functions of sleep 6
1.2. The immune system and its relationship to sleep 7
1.3. Wound healing and its relationship to sleep 10
1.4. Caenorhabditis elegans is a well-studied model organism 12
1.4.1. Sleep in C. elegans 15
1.4.2. The C. elegans cuticle 18
1.4.3. Immunity in C. elegans 19
1.4.4. Wound healing response in C. elegans 22
2. Previous results 25
2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25
2.2. NAS-38 increases sleep mostly through the RIS neuron 25
2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25
2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27
2.5. Overexpression of AMPs induces RIS dependent quiescence 30
2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31
3. Thesis Aims 34
3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34
3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35
4. Materials and Methods 36
4.1. C. elegans maintenance 36
4.2. C. elegans crossing and genotyping 41
4.3. Creation of transgenic animals 45
4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45
4.3.2. Microparticle bombardment 45
4.3.3. CRISPR/Cas9 system 46
4.4. Synchronizing worm cultures by hypochlorite treatment 48
4.5. Imaging 49
4.5.1. Imaging setups 49
4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50
4.5.2.1. Imaging of heterozygous mutants 50
4.5.3. DIC imaging in the temperature control device 51
4.5.4. Fluorescent imaging experiments 51
4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51
4.5.4.2. nlp-29p::GFP in L4 larvae 52
4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52
4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52
4.5.4.5. Functional Ca2+ imaging in young adults 52
4.5.4.6. Fluorescence imaging across the whole developmental time 54
4.5.4.7. Nuclear decompaction assays 55
4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55
4.5.4.9. Imaging DPY-13::mKate2 in young adults 56
4.6. Image analysis 56
4.6.1. Assessment of developmental time and lethargus detection 56
4.6.2. Sleep detection in DIC mode 56
4.6.3. Analyzing functional Ca2+ images 57
4.6.4. Fluorescent reporter analysis during long-term imaging 57
4.7. RNAi-by-feeding 58
4.8. Transcriptome analysis 59
4.8.1. Analysis of the nas-38(ok3407) transcriptome 59
4.8.2. Analysis of the wounding transcriptome 59
4.9. Epidermal wounding 62
4.9.1. Laser wounding 62
4.9.2. Needle wounding 62
4.9.3. Survival assay 63
4.10. Scanning Electron Microscopy (SEM) 63
4.11. Histamine-inducible hyperpolarization of RIS 64
4.12. Cuticle integrity test with Sodium hypochlorite 64
4.13. NPR-12 receptor modeling 64
4.14. Quantification and statistical analysis 65
5. Results 66
5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66
5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66
5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66
5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69
5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72
5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75
5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77
5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80
5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81
5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83
5.1.10. AMPs contribute to the survival after wounding 85
5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87
5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87
5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87
5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89
5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95
5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101
5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106
5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109
5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114
5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120
5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122
6. Discussion and Outlook 128
6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128
6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130
6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131
6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132
6.5. Genetic sleep deprivation alters cuticle formation 135
6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137
6.7. Conclusion 139
7. References 140
8. Acknowledgements 163
9. Appendix 166
9.1. Standard reagents 166
9.2. Sequence summary of PHX3754 167
9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171
9.4. Permissions to reprint figures 174
9.5. Experimental author contributions 175
9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176
9.7. Overlap of the adult wounding transcriptome with other data sets 179
9.8. Curriculum Vitae – Marina Patricia Sinner 181Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a protective response. This hypothesis, however, was hitherto lacking final evidence and pathway components.
Research questions and hypotheses: I aimed to characterize the molecular mechanism by which separate triggers of innate immunity, i. e. NAS-38 and wounding, induce sleep. I specifically addressed two questions: Firstly, which domains of the NAS-38 protein are involved in sleep regulation? As the astacin domain is predicted to be the active protease domain of NAS-38, I expected a role for it also in sleep induction by NAS-38. Secondly, what is the role of AMPs in signaling immunity-induced sleep? As they have been shown to be upregulated during times of increased sleep in the nas-38 mutant and after wounding, I expected AMPs to be involved in signaling sleep from the epidermis to the nervous system. In a second step, I investigated the molecular mechanisms underlying the benefits of sleep for surviving injury. Again, I addressed two questions: Firstly, does genetic sleep deprivation alter the transcriptional wounding response? As sleep has a role in many fundamental processes and sleeplessness increases mortality upon wounding, I hypothesized that genetic sleep deprivation impairs wounding-induced changes of transcriptional activity. Secondly, does sleep help building robustness before encountering injury? During larval development the synthesis of a new cuticle coincides with sleep. Thus, I hypothesized that genetic sleep deprivation impairs proper cuticle formation. Methods: To dissect the signaling mechanisms by which NAS-38 and wounding induced sleep, I followed sleep behavior of C. elegans by long-term imaging in agarose microchambers. I performed a structure-function analysis with different nas-38 mutants, each carrying a deletion of a different domain. Moreover, I screened for suppressors of sleep induced by NAS 38 or wounding. To test for redundancy of the AMP family, I investigated the suppression-phenotype of a CRISPR/Cas9 edited multi-knockout mutant lacking 19 AMPs. To identify downstream effectors of the AMP NLP 29, I induced sleep by overexpressing NLP 29 from a heat-shock promoter and analyzed the suppression-phenotype of different knockout mutants. For the second project, I addressed the question how sleep aids recovery from injury. I followed fluorescent reporters of previously described wounding response pathways by fluorescent long-term imaging in wild-type and genetically sleep-deprived animals. Moreover, I compared the transcriptomes of adult wild-type and genetically sleep-deprived worms both wounded and unwounded. To investigate the structure of the cuticle, I analyzed scanning electron microscopy images. Results: In the first project, I could show that NAS-38 indeed increases sleep via its astacin domain in a process that is modulated by the TSP-1 domain. Moreover, I could show that many AMPs act redundantly in mediating immunity-induced sleep downstream of NAS-38 and after wounding. I demonstrated that the AMP NLP-29 signals sleep via the neuropeptide receptor NPR 12. This receptor can mediate sleep when it is specifically expressed in command interneurons of a circuit that has been shown to activate RIS. Interestingly, I also found that EGFR signaling is required to mediate NLP-29-induced sleep.
In the second project, I found that sleeplessness does not dramatically alter the transcriptional wounding response. However, I could show that transcription is altered already in the unwounded non-sleeping mutant. This affects, among others, a specific subset of oscillating collagen-coding genes, whose expression usually peaks around the end of lethargus. As the timing of expression of collagens is thought to be highly important for proper cuticle formation, I characterized the cuticle of the aptf-1(gk794) mutant. I could show that young adult aptf 1(gk794) worms indeed have a structural defect affecting cuticular furrows in the region adjacent to the alae, which could potentially decrease specific aspects of resilience of the cuticle. Thus, sleep might be required to build robustness in the form of a properly structured cuticle. Conclusion: In this PhD project, I completed the characterization of a novel mechanism by which wounding signals sleep from the periphery to the nervous system as part of the immune response in C. elegans. I could show that AMPs act as cross-tissue signals from the epidermis to a neuronal RIS-controlling circuit that ultimately leads to sleep induction. As components of this molecular pathway are highly conserved, AMPs might also induce sleep to promote recovery from injury in other organisms, including humans. Moreover, I laid the foundations for dissecting the molecular mechanisms behind the functions of sleep for healing and survival. Even though the disability to sleep did not seem to drastically change the transcriptional response to wounding, my results indicate a role for
Neural circuit architecture and evolutionary adaptations in the Drosophila olfactory system
This thesis aimed to understand the details of the neural architecture of the olfactory system in flies of the genus Drosophila and its evolutionary adaptation to diverse species-specific environmental conditions. In the insect olfactory first relay station, the antennal lobe, olfactory sensory neurons (OSNs) relay odorant previously induced signals to segregated coding units, the olfactory glomeruli. At this level, the system already extracts and encodes valuable information, such as odor identity, concentration, or odor location. Most insect’s volatile odorants are encoded in a combinatorial manner, i.e. one odorant activates many distinct glomerular coding units, which in turn are activated by several odorants. Selected odorants with particular ecological importance are encoded in single and distinct glomerular circuitries. In the first part of the thesis, I show in a dense connectome analysis in the fruit fly, Drosophila melanogaster, that these dedicated glomerular coding units have evolved specific circuit features that might be important to ensure improved accuracy and signal amplification in a single glomerular system. This thesis discovered furthermore a substantial amount of autapses, self-activating feedback synapses, along the large dendrite of a uniglomerular projection neuron, which are likely to play a role in neuronal communication and/or modulation. In the second part, I discovered deep invaginations nearby presynaptic sites of mainly OSNs formed by protrusions from neighboring neurons. The so-called “synaptic spinules” play a role in neuronal communication and/ or modulation. In the last part of my thesis, I contributed to a large-scale analysis of over 60 species of the genus Drosophila with respect to their phenotypic properties, behavior, and their olfactory and visual systems. This work provides important information on synaptic circuits and architecture and on the question, of how the system might has evolved in the best possible way
MicroRNAs and GRK2 as modulators of Kiss1/GPR54 system: Physiopathological role in pubertal alterations and obesity induced hypogonadism
The reproductive function is governed by the so-called hypothalamic-pituitary-gonadal (HPG) axis, where an intricated network of central, peripheral and external factors determine hormonal balance and proper functioning of the reproductive system and the gonadal function, guaranteeing the perpetuation of the species 1–3. In recent years, it has been documented that a plethora of central (glutamate, GABA, NKB, NPY) 4–7, peripheral (insulin, leptin, ghrelin) 8,9 and external (nutritional availability, endocrine disruptors, circadian rhythms) 10–13 cues converge (either acting directly or indirectly) onto Kiss1 neurons in the hypothalamus, as major signaling hub of the HPG axis 14, whose products, kisspeptins, act on the GnRH neurons, via its canonical receptor, GPR5415, activating puberty onset and reproductive function. In addition, it is well recognized that reproductive function is altered under conditions of metabolic distress, ranging from subnutrition to obesity, type 2 diabetes and metabolic syndrome, which are bound to numerous perturbations, including disordered puberty, central hypogonadism (mainly in males) and cardiometabolic impairment16,17.
MicroRNAs have been recently pointed out as essential players in the control of normal pubertal development18–21, although no study has addressed the specific regulation of Kiss1, at central levels, exerted by miRNAs 21,22. Further, the effects of miRNAs in the pathogenesis of central hypogonadism are completely unexplored. In parallel, the Kiss1/GPR54 system is a key element for the integration of the energetic status and reproductive capacity 23, where GPR54 inactivating mutations were described decades ago as underlying origin of hypogonadotropic hypogonadism 24,25. The G-protein coupled receptor kinase 2 (GRK2) 26, which is largely recognized as pleiotropic regulator of cellular signaling 27–29, has been suggested in vitro as a modulator of GPR5426. Nevertheless, no studies had addressed to date its potential roles in proper pubertal development and maintenance of reproductive capacity.
In the above context, this Doctoral Thesis has addressed, as main aims, (i) the putative role of specific miRNAs in the physiological control of puberty via regulation of the Kiss1 system; (ii) the pathophysiological role of miRNAs in obesity-induced hypogonadism (OIH), their interplay with Kiss1 and their potential therapeutic implications; (iii) the role of GRK2 in the control of puberty and the HPG axis through regulation of GPR54 in normal conditions and under nutritional stress; and (iv) the implication of GRK2 in OIH through GPR54 regulation.La función reproductora está determinada por el correcto funcionamiento del eje hipotálamohipofiso-gonadal (HHG), donde una compleja red de factores centrales, periféricos y externos determinan el balance hormonal necesario para la adquisición de la capacidad reproductora y, en consecuencia, para el mantenimiento de las especies 1–3. De este modo, se ha documentado que una multitud de factores centrales (GABA, glutamato, NKB, NPY) 4–7, periféricos (leptina, insulina o ghrelina) 8,9 y externos (disruptores endocrinos, aporte energético, ritmos circadianos) 10–13 convergen (actuando directa o indirectamente) en las neuronas Kiss1 hipotalámicas como principal núcleo del eje HHG 14 y cuyo producto, las kisspeptinas, transmitirán información a la neurona GnRH, por medio de su receptor canónico, GPR5415 , activando la pubertad y la función reproductora. Al mismo tiempo, existen evidencias sobre la afectación de la función reproductora como consecuencia del desequilibrio homeostático presente en situaciones como la subnutrición o la obesidad, diabetes mellitus tipo 2 o síndrome metabólico, que se encuentran vinculadas a numerosos desórdenes, incluyendo alteraciones de la edad de pubertad, el hipogonadismo central (principalmente masculino) y la enfermedad cardiovascular 16,17. Datos recientes señalan que los microRNAs (miRNAs) son elementos implicados en la correcta transición puberal 18–21, aunque existen pocos estudios dirigidos a evaluar al papel de los miRNAs en la regulación específica de la expresión de Kiss1, a nivel central19,22. Además, la desregulación de determinados miRNAs en condiciones de hipogonadismo central, su impacto sobre el sistema Kiss1 y su implicación fisiopatológica en esta condición, permanecen inexplorados. En paralelo, el sistema Kiss1/GPR54 es un elemento clave en la integración del estado energético y la capacidad reproductora23, estando descrito que mutaciones inactivantes en GPR54 son una causa subyacente en determinados casos de hipogonadismo hipogonadotropo 24,25. La quinasa de receptor acoplado a proteína G (GRK2) 26 está reconocida como un regulador pleiotrópico de la señalización celular 27–29 y ha sido demostrada su capacidad para regular GPR54, in vitro 26. En cualquier caso, no existen estudios relacionados con su potencial implicación en la correcta maduración puberal y en el mantenimiento de la capacidad reproductora in vivo.
En base a lo anterior, esta Tesis Doctoral ha abordado, como objetivos principales, (i) el estudio del papel específico de microRNAs en la regulación puberal a través de la regulación del sistema Kiss1; (ii) el papel fisiopatológico de los microRNAs en el hipogonadismo central inducido por obesidad (HIO) y sus potenciales implicaciones terapéuticas; (iii) el papel de GRK2 en el control de la puberal y el eje HHG mediante la regulación de GPR54 en condiciones control y de estrés nutricional; y (iv) la implicación de GRK2 en HIO a través de la regulación de GPR54
Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy
Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.P 28338 - Austrian Science Fund FWF; U01 MH105971 - NIMH NIH HHS; U01 MH114824 - NIMH NIH HHS; Howard Hughes Medical InstituteAccepted manuscrip
Sleep is required to consolidate odor memory and remodel olfactory synapses
Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here, we show that, although the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear if, in any system, sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures
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