CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters.

Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.

Glutamate and clock help bean bugs track seasonal reproductive changes.

Animals adapt their seasonal physiology by measuring photoperiodic changes over the calendar year. A new study in PLOS Biology uncovers changes in glutamate dynamics in the bean bug that are dependent on photoperiod and a clock gene.

Integration of photoperiodic and temperature cues by the circadian clock to regulate insect seasonal adaptations.

Organisms adapt to unfavorable seasonal conditions to survive. These seasonal adaptations rely on the correct interpretation of environmental cues such as photoperiod, and temperature. Genetic studies in several organisms, including the genetic powerhouse Drosophila melanogaster, indicate that circadian clock components, such as period and timeless, are involved in photoperiodic-dependent seasonal adaptations, but our understanding of this process is far from complete. In particular, the role of temperature as a key factor to complement photoperiodic response is not well understood. The development of new sequencing technologies has proven extremely useful in understanding the plastic changes that the clock and other cellular components undergo in different environmental conditions, including changes in gene expression and alternative splicing. This article discusses the integration of photoperiod and temperature for seasonal biology as well as downstream molecular and cellular pathways involved in the regulation of physiological adaptations that occur with changing seasons. We focus our discussion on the current understanding of the involvement of the molecular clock and the circadian clock neuronal circuits in these adaptations in D. melanogaster.

Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila.

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

Study of the release of endogenous amines in Drosophila brain in vivo in response to stimuli linked to aversive olfactory conditioning.

A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. Here, we report a procedure to record for the first time the release of endogenous amines -dopamine, serotonin and octopamine- in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, we carried out recordings in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, we detect an increase in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and shed new light on the contribution of these amines to olfactory processing in this animal model.

The Drosophila ortholog of the schizophrenia-associated CACNA1A and CACNA1B voltage-gated calcium channels regulate memory, sleep and circadian rhythms.

Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.

Characterization of a presymptomatic stage in a Drosophila Parkinson's disease model: Unveiling dopaminergic compensatory mechanisms.

Parkinson's disease (PD) is a degenerative disorder characterized by several motor symptoms including shaking, rigidity, slow movement and difficult walking, which has been associated to the death of nigro-striatal dopaminergic neurons. >90% of PD patients also present olfactory dysfunction. Although the molecular mechanisms responsible for this disease are not clear, hereditary PD is linked to mutations in specific genes, including the PTEN-induced putative kinase 1 (PINK1). In this work we provide for the first time a thorough temporal description of the behavioral effects induced by a mutation in the PINK1 gene in adult Drosophila, a previously described animal model for PD. Our data suggests that the motor deficits associated to PD are fully revealed only by the third week of age. However, olfactory dysfunction is detected as early as the first week of age. We also provide immunofluorescence and neurochemical data that let us propose for the first time the idea that compensatory changes occur in this Drosophila model for PD. These compensatory changes are associated to specific components of the dopaminergic system: the biosynthetic enzymes, Tyrosine hydroxylase and Dopa decarboxylase, and the Dopamine transporter, a plasma membrane protein involved in maintaining dopamine extracellular levels at physiologically relevant levels. Thus, our behavioral, immunofluorescence and neurochemical data help define for the first time presymptomatic and symptomatic phases in this PD animal model, and that compensatory changes occur in the dopaminergic neurons in the presymptomatic stage.

Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes.

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

CRUMB: a shiny-based app to analyze rhythmic feeding in Drosophila using the FLIC system.

Rhythmic feeding activity has become an important research area for circadian biologists as it is now clear that metabolic input is critical for regulating circadian rhythms, and chrononutrition has been shown to promote health span. In contrast to locomotor activity rhythm, studies conducting high throughput analysis of Drosophila rhythmic food intake have been limited and few monitoring system options are available. One monitoring system, the Fly Liquid-Food Interaction Counter (FLIC) has become popular, but there is a lack of efficient analysis toolkits to facilitate scalability and ensure reproducibility by using unified parameters for data analysis. Here, we developed Circadian Rhythm Using Mealtime Behavior (CRUMB), a user-friendly Shiny app to analyze data collected using the FLIC system. CRUMB leverages the 'plotly' and 'DT' packages to enable interactive raw data review as well as the generation of easily manipulable graphs and data tables. We used the main features of the FLIC master code provided with the system to retrieve feeding events and provide a simplified pipeline to conduct circadian analysis. We also replaced the use of base functions in time-consuming processes such as 'rle' and 'read.csv' with faster versions available from other packages to optimize computing time. We expect CRUMB to facilitate analysis of feeding-fasting rhythm as a robust output of the circadian clock.

Seasonal cues act through the circadian clock and pigment-dispersing factor to control EYES ABSENT and downstream physiological changes.

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain to alter seasonal biology are poorly understood. We previously reported that EYES ABSENT (EYA) shows higher levels in cold temperature or short photoperiod and promotes winter physiology in Drosophila. Nevertheless, how EYA senses seasonal cues is unclear. Pigment-dispersing factor (PDF) is a neuropeptide important for regulating circadian output rhythms. Interestingly, PDF has also been shown to regulate seasonality, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of EYA in mediating the function of PDF on seasonal biology. We observed that PDF abundance is lower on cold and short days as compared with warm and long days, contrary to what was previously observed for EYA. We observed that manipulating PDF signaling in eya+ fly brain neurons, where EYA and PDF receptor are co-expressed, modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and EYA-independent mechanisms. At the molecular level, altering PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA promoting its degradation, thus explaining the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

An Early Disturbance in Serotonergic Neurotransmission Contributes to the Onset of Parkinsonian Phenotypes in Drosophila melanogaster.

Parkinson's disease (PD) is a neurodegenerative disease characterized by motor symptoms and dopaminergic cell loss. A pre-symptomatic phase characterized by non-motor symptoms precedes the onset of motor alterations. Two recent PET studies in human carriers of mutations associated with familial PD demonstrate an early serotonergic commitment-alteration in SERT binding-before any dopaminergic or motor dysfunction, that is, at putative PD pre-symptomatic stages. These findings support the hypothesis that early alterations in the serotonergic system could contribute to the progression of PD, an idea difficult to be tested in humans. Here, we study some components of the serotonergic system during the pre-symptomatic phase in a well-characterized Drosophila PD model, Pink1B9 mutant flies. We detected lower brain serotonin content in Pink1B9 flies, accompanied by reduced activity of SERT before the onset of motor dysfunctions. We also explored the consequences of a brief early manipulation of the serotonergic system in the development of motor symptoms later in aged animals. Feeding young Pink1B9 flies with fluoxetine, a SERT blocker, prevents the loss of dopaminergic neurons and ameliorates motor impairment observed in aged mutant flies. Surprisingly, the same pharmacological manipulation in young control flies results in aged animals exhibiting a PD-like phenotype. Our findings support that an early dysfunction in the serotonergic system precedes and contributes to the onset of the Parkinsonian phenotype in Drosophila.

Assessing olfactory, memory, social and circadian phenotypes associated with schizophrenia in a genetic model based on Rim.

Schizophrenia shows high heritability and several of the genes associated with this disorder are involved in calcium (Ca2+) signalling and synaptic function. One of these is the Rab-3 interacting molecule-1 (RIM1), which has recently been associated with schizophrenia by Genome Wide Association Studies (GWAS). However, its contribution to the pathophysiology of this disorder remains unexplored. In this work, we use Drosophila mutants of the orthologue of RIM1, Rim, to model some aspects of the classical and non-classical symptoms of schizophrenia. Rim mutants showed several behavioural features relevant to schizophrenia including social distancing and altered olfactory processing. These defects were accompanied by reduced evoked Ca2+ influx and structural changes in the presynaptic terminals sent by the primary olfactory neurons to higher processing centres. In contrast, expression of Rim-RNAi in the mushroom bodies (MBs), the main memory centre in flies, spared learning and memory suggesting a differential role of Rim in different synapses. Circadian deficits have been reported in schizophrenia. We observed circadian locomotor activity deficits in Rim mutants, revealing a role of Rim in the pacemaker ventral lateral clock neurons (LNvs). These changes were accompanied by impaired day/night remodelling of dorsal terminal synapses from a subpopulation of LNvs and impaired day/night release of the circadian neuropeptide pigment dispersing factor (PDF) from these terminals. Lastly, treatment with the commonly used antipsychotic haloperidol rescued Rim locomotor deficits to wildtype. This work characterises the role of Rim in synaptic functions underlying behaviours disrupted in schizophrenia.

Neonicotinoids disrupt memory, circadian behaviour and sleep.

Globally, neonicotinoids are the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects. Neonicotinoids are nicotinic acetylcholine receptor agonists. Memory, circadian rhythmicity and sleep are essential for efficient foraging and pollination and require nicotinic acetylcholine receptor signalling. The effect of field-relevant concentrations of the European Union-banned neonicotinoids: imidacloprid, clothianidin, thiamethoxam and thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin prevented the day/night remodelling and accumulation of pigment dispersing factor (PDF) neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dß2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or sleep/circadian behaviour respectively. Disruption of learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.

Dop1R1, a type 1 dopaminergic receptor expressed in Mushroom Bodies, modulates Drosophila larval locomotion.

As in vertebrates, dopaminergic neural systems are key regulators of motor programs in insects, including the fly Drosophila melanogaster. Dopaminergic systems innervate the Mushroom Bodies (MB), an important association area in the insect brain primarily associated to olfactory learning and memory, but that has been also implicated with the execution of motor programs. The main objectives of this work is to assess the idea that dopaminergic systems contribute to the execution of motor programs in Drosophila larvae, and then, to evaluate the contribution of specific dopaminergic receptors expressed in MB to these programs. Our results show that animals bearing a mutation in the dopamine transporter show reduced locomotion, while mutants for the dopaminergic biosynthetic enzymes or the dopamine receptor Dop1R1 exhibit increased locomotion. Pan-neuronal expression of an RNAi for the Dop1R1 confirmed these results. Further studies show that animals expressing the RNAi for Dop1R1 in the entire MB neuronal population or only in the MB γ-lobe forming neurons, exhibit an increased motor output, as well. Interestingly, our results also suggest that other dopaminergic receptors do not contribute to larval motor behavior. Thus, our data support the proposition that CNS dopamine systems innervating MB neurons modulate larval locomotion and that Dop1R1 mediates this effect.

The behavioral and neurochemical characterization of a Drosophila dysbindin mutant supports the contribution of serotonin to schizophrenia negative symptoms.

Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. Here we evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. We also report a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. Our data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, our data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. We argue that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease.

Restoration of Olfactory Memory in Drosophila Overexpressing Human Alzheimer's Disease Associated Tau by Manipulation of L-Type Ca2+ Channels.

The cellular underpinnings of memory deficits in Alzheimer's disease (AD) are poorly understood. We utilized the tractable neural circuits sub-serving memory in Drosophila to investigate the role of impaired Ca2+ handling in memory deficits caused by expression of human 0N4R isoform of tau which is associated with AD. Expression of tau in mushroom body neuropils, or a subset of mushroom body output neurons, led to impaired memory. By using the Ca2+ reporter GCaMP6f, we observed changes in Ca2+ signaling when tau was expressed in these neurons, an effect that could be blocked by the L-type Ca2+ channel antagonist nimodipine or reversed by RNAi knock-down of the L-type channel gene. The L-type Ca2+ channel itself is required for memory formation, however, RNAi knock-down of the L-type Ca2+ channel in neurons overexpressing human tau resulted in flies whose memory is restored to levels equivalent to wild-type. Expression data suggest that Drosophila L-type Ca2+ channel mRNA levels are increased upon tau expression in neurons, thus contributing to the effects observed on memory and intracellular Ca2+ homeostasis. Together, our Ca2+ imaging and memory experiments suggest that expression of the 0N4R isoform of human tau increases the number of L-type Ca2+ channels in the membrane resulting in changes in neuronal excitability that can be ameliorated by RNAi knockdown or pharmacological blockade of L-type Ca2+ channels. This highlights a role for L-type Ca2+ channels in tauopathy and their potential as a therapeutic target for AD.

Characterization of a Novel Drosophila SERT Mutant: Insights on the Contribution of the Serotonin Neural System to Behaviors.

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. Here we decided to advance our understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, we characterized for the first time a mutant for Drosophila SERT (dSERT) and additionally used a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), whose mechanism of action involves the SERT protein. Our results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. We also show that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. Our neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. Our in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, our neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.

A stigmatic ultraviolet-visible monochromator for use with a high brightness laser driven plasma light source.

Laser driven plasma light sources offer highly intense output in the UV-visible region combined with a source size as small as 100 µm. In order to effectively use the small source size in high brightness applications, a stigmatic monochromator and focusing system must be used. Here we describe a simple brightness preserving optical system that should be useful across a broad range of applications. The output flux of this system is between 6 × 10(11) ph∕s and 4 × 10(12) ph∕s with a spectra resolution of 1.7 nm and field spot size of 0.1 mm from the UV to the VIS spectra range.