Sleep-promoting neurons remodel their response properties to calibrate sleep drive with environmental demands

Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal fan-shaped body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation) in Drosophila. We report that dFB neurons change their physiological response-properties to dopamine (DA) and allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together, these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism

The role of cryptochrome in the drosophila circadian clock

The circadian clock of Drosophila melanogaster consists of at least two interlocked feedback loops. In the first, the period and timeless gene products negatively regulate their own transcription. CRYPTOCHROME (CRY) is the dedicated circadian photoreceptor, and flies carrying a strong hypomorphic mutation in the cry gene have severely blunted circadian photoresponses. CRY physically interacts with the core components of the clock, PERIOD (PER) and TIMELESS (TIM) in a light-dependent manner. Previous work carried out in the laboratory showed that removing 20 amino acids at the C-terminus of CRY to create CRYDelta results in the loss of light-dependency of CRY interactions in yeast two-hybrid assays. Based on this work, the aim of my project was to study the role of the CRY C-terminus in vivo by clock neurons targeted overexpression of CRYDelta with the hypothesis that it should behave as a constitutively active form of the protein. CRYDelta flies have long period of locomotor activity in constant darkness, show abnormal responses to light and exhibit altered oscillation of the PER and TIM proteins in central and peripheral clocks. These phenotypes are reminiscent of responses observed when wild-type flies are kept under continuous low-light intensity. Therefore, this study provides strong behavioural, molecular and immunohistochemical evidence confirming that CRYDelta is constitutively active, and elicits continuous light responses. Moreover, previous work demonstrated that CRY role in the Drosophila clock exclusively involves light signalling to the core components of the clock. This study identified a potential new light-dependent function for CRY in vivo.

Drosophila model systems for genetic sleep research

The successful sequencing of the human genome in 2000, along with sequencing the genomes of several genetic model organisms, have ushered in a new era of sleep research in which the power of genetic and genomic strategies can be brought to bear on previously intractable problems pertaining to sleep mechanism and function. Indeed, genetic studies in humans (Chapter 14), mice (Chapter 15), zebra fish (Chapter 6), Drosophila melanogaster (Chapter 5), and Caenorhabditis elegans (Chapter 6) have been successful in advancing our understanding of basic sleep mechanisms (see [1]). Although it is still common for laboratories to focus their research on a preferred model organism, it is becoming increasingly clear that progress can be enhanced when labs cross-validate their findings between more than one species [2–6]. This is particularly true for human studies in which establishing causation is non-trivial. For example, He and colleagues in 2009 identified a point mutation in human DEC2 that was associated with a short-sleeping phenotype and demonstrated causation using transgenic mice and flies [4]. Similarly, in 2013, Allebrandt et al. identified a variant of the ABCC9 gene in a human genome-wide association study (GWAS) and then, using Drosophila genetics, demonstrated a functional role for ABCC9 gene in regulating sleep time [6]. Likewise, Freeman and colleagues used Drosophila genetics to show that a gene identified in human GWAS studies for restless legs syndrome (RLS), BTB9, recapitulates key aspects of RLS in flies [5]. With this in mind, the following chapter will review the use of genetics in Drosophila to advance sleep research. Our goal is to highlight the sophisticated genetic toolbox that allows studies to be conducted quickly and at little cost. Together, these data demonstrate that the fly is well suited for not only revealing basic sleep mechanisms in its own right, but can also be directly incorporated into human studies to provide additional insight into causative mechanisms

Learning and memory: do bees dream?

In mammals, evidence for memory reactivation during sleep highlighted the important role that sleep plays in memory consolidation. A new study reports that memory reactivation is evolutionarily conserved and can also be found in the honeybee

Identification of Genes that Maintain Behavioral and Structural Plasticity during Sleep Loss

Although patients with primary insomnia experience sleep disruption, they are able to maintain normal performance on a variety of cognitive tasks. This observation suggests that insomnia may be a condition where predisposing factors simultaneously increase the risk for insomnia and also mitigate against the deleterious consequences of waking. To gain insight into processes that might regulate sleep and buffer neuronal circuits during sleep loss, we manipulated three genes, fat facet (faf), highwire (hiw) and the GABA receptor Resistance to dieldrin (Rdl), that were differentially modulated in a Drosophila model of insomnia. Our results indicate that increasing faf and decreasing hiw or Rdl within wake-promoting large ventral lateral clock neurons (lLNvs) induces sleep loss. As expected, sleep loss induced by decreasing hiw in the lLNvs results in deficits in short-term memory and increases of synaptic growth. However, sleep loss induced by knocking down Rdl in the lLNvs protects flies from sleep-loss induced deficits in short-term memory and increases in synaptic markers. Surprisingly, decreasing hiw and Rdl within the Mushroom Bodies (MBs) protects against the negative effects of sleep deprivation (SD) as indicated by the absence of a subsequent homeostatic response, or deficits in short-term memory. Together these results indicate that specific genes are able to disrupt sleep and protect against the negative consequences of waking in a circuit dependent manner

Antimicrobial peptides modulate long-term memory.

Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory we performed mRNA sequencing from single Drosophila heads following behavioral training that produces long-lasting memory. Surprisingly, we found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system

Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

The Drosophila melanogaster circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light–dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cryb mutation reduces the light sensitivity of the fly’s clock, yet locomotor activity rhythms in constant darkness or light–dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when we combined a period-null mutation with cryb, circadian rhythmicity in locomotor behavior in light–dark cycles, as measured by a number of different criteria, was restored. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and TIM protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, we have uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although we do not yet have a molecular correlate for these apparently iconoclastic observations, we provide a systems explanation for these results based on differential sensitivities of subsets of circadian pacemaker neurons to light

Enhanced sleep reverses memory deficits and underlying pathology in drosophila models of Alzheimer's disease

To test the hypothesis that sleep can reverse cognitive impairment during Alzheimer's disease, we enhanced sleep in flies either co-expressing human amyloid precursor protein and Beta-secretase (APP:BACE), or in flies expressing human tau. The ubiquitous expression of APP:BACE or human tau disrupted sleep. The sleep deficits could be reversed and sleep could be enhanced when flies were administered the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Expressing APP:BACE disrupted both Short-term memory (STM) and Long-term memory (LTM) as assessed using Aversive Phototaxic Suppression (APS) and courtship conditioning. Flies expressing APP:BACE also showed reduced levels of the synaptic protein discs large (DLG). Enhancing sleep in memory-impaired APP:BACE flies fully restored both STM and LTM and restored DLG levels. Sleep also restored STM to flies expressing human tau. Using live-brain imaging of individual clock neurons expressing both tau and the cAMP sensor Epac1-camps, we found that tau disrupted cAMP signaling. Importantly, enhancing sleep in flies expressing human tau restored proper cAMP signaling. Thus, we demonstrate that sleep can be used as a therapeutic to reverse deficits that accrue during the expression of toxic peptides associated with Alzheimer's disease