Mating disrupts morning anticipation in Drosophila melanogaster females.

After mating, the physiology of Drosophila females undergo several important changes, some of which are reflected in their rest-activity cycles. To explore the hypothesis that mating modifies the temporal organization of locomotor activity patterns, we recorded fly activity by a video tracking method. Monitoring rest-activity patterns under light/dark (LD) cycles indicated that mated females lose their ability to anticipate the night-day transition, in stark contrast to males and virgins. This postmating response is mediated by the activation of the sex peptide receptor (SPR) mainly on pickpocket (ppk) expressing neurons, since reducing expression of this receptor in these neurons restores the ability to anticipate the LD transition in mated females. Furthermore, we provide evidence of connectivity between ppk+ neurons and the pigment-dispersing factor (PDF)-positive ventral lateral neurons (sLNv), which play a central role in the temporal organization of daily activity. Since PDF has been associated to the generation of the morning activity peak, we hypothesized that the mating signal could modulate PDF levels. Indeed, we confirm that mated females have reduced PDF levels at the dorsal protocerebrum; moreover, SPR downregulation in ppk+ neurons mimics PDF levels observed in males. In sum, our results are consistent with a model whereby mating-triggered signals reach clock neurons in the fly central nervous system to modulate the temporal organization of circadian behavior according to the needs of the new status.

High-Frequency Neuronal Bursting is Essential for Circadian and Sleep Behaviors in Drosophila.

Circadian rhythms have been extensively studied in Drosophila; however, still little is known about how the electrical properties of clock neurons are specified. We have performed a behavioral genetic screen through the downregulation of candidate ion channels in the lateral ventral neurons (LNvs) and show that the hyperpolarization-activated cation current Ih is important for the behaviors that the LNvs influence: temporal organization of locomotor activity, analyzed in males, and sleep, analyzed in females. Using whole-cell patch clamp electrophysiology we demonstrate that small LNvs (sLNvs) are bursting neurons, and that Ih is necessary to achieve the high-frequency bursting firing pattern characteristic of both types of LNvs in females. Since firing in bursts has been associated to neuropeptide release, we hypothesized that Ih would be important for LNvs communication. Indeed, herein we demonstrate that Ih is fundamental for the recruitment of pigment dispersing factor (PDF) filled dense core vesicles (DCVs) to the terminals at the dorsal protocerebrum and for their timed release, and hence for the temporal coordination of circadian behaviors.SIGNIFICANCE STATEMENT Ion channels are transmembrane proteins with selective permeability to specific charged particles. The rich repertoire of parameters that may gate their opening state, such as voltage-sensitivity, modulation by second messengers and specific kinetics, make this protein family a determinant of neuronal identity. Ion channel structure is evolutionary conserved between vertebrates and invertebrates, making any discovery easily translatable. Through a screen to uncover ion channels with roles in circadian rhythms, we have identified the Ih channel as an important player in a subset of clock neurons of the fruit fly. We show that lateral ventral neurons (LNvs) need Ih to fire action potentials in a high-frequency bursting mode and that this is important for peptide transport and the control of behavior.

Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons.

We have previously reported that pigment dispersing factor (PDF) neurons, which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections, a phenomenon called circadian structural plasticity. Axonal arborizations display higher complexity during the day and become simpler at night, and this remodeling involves changes in the degree of connectivity. This phenomenon depends on the clock present within the ventrolateral neurons (LNvs) as well as in glia. In this work, we characterize in detail the contribution of the PDF neuropeptide to structural plasticity at different times across the day. Using diverse genetic strategies to temporally restrict its downregulation, we demonstrate that even subtle alterations to PDF cycling at the dorsal protocerebrum correlate with impaired remodeling, underscoring its relevance for the characteristic morning spread; PDF released from the small LNvs (sLNvs) and the large LNvs (lLNvs) contribute to the process. Moreover, forced depolarization recruits activity-dependent mechanisms to mediate growth only at night, overcoming the restriction imposed by the clock on membrane excitability. Interestingly, the active process of terminal remodeling requires PDF receptor (PDFR) signaling acting locally through the cyclic-nucleotide-gated channel ion channel subunit A (CNGA). Thus, clock-dependent PDF signaling shapes the connectivity of these essential clock neurons on daily basis.

Circadian pacemaker neurons change synaptic contacts across the day.

Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters [1, 2]. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity [3-7]. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers [8]. Here we show that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, we found that the degree of daily structural plasticity exceeds that originally described [8], underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique [9], we showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead us to propose that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels.

Circadian period integrates network information through activation of the BMP signaling pathway.

Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

A role for the membrane protein M6 in the Drosophila visual system.

BACKGROUND: Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system. RESULTS: The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light. CONCLUSIONS: Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

Adult-specific electrical silencing of pacemaker neurons uncouples molecular clock from circadian outputs.

BACKGROUND: Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral lateral neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. RESULTS: Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PERIOD (PER) oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. CONCLUSIONS: Although electrical activity of the sLNvs is not a clock component, it coordinates circuit outputs leading to rhythmic behavior.

Circadian plasticity: from structure to behavior.

Over the years it has become clear that the biological clock acts at different levels, ranging from the control of gene expression, protein stability, or subcellular localization of key proteins, to the fine tuning of network properties and modulation of input signals, ultimately ensuring that the organism will be best synchronized to a changing environment at the physiological and behavioral levels. The purpose of this chapter is to discuss the circadian control of clock outputs, spanning the most immediate ones within pacemaker neurons (i.e., membrane excitability, release of neurotransmitters, structural changes) to the circadian modulation of different behaviors (locomotor activity, learning and memory, social interaction), with a focus on the examples that shed light on the surprising degree of plasticity that characterizes the underlying circuits.

M6 membrane protein plays an essential role in Drosophila oogenesis.

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.

A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.

Circadian remodeling of neuronal circuits involved in rhythmic behavior.

Clock output pathways are central to convey timing information from the circadian clock to a diversity of physiological systems, ranging from cell-autonomous processes to behavior. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is unclear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the time of day information from core pacemaker cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Here we describe a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit, which display higher complexity during the day and significantly lower complexity at nighttime, both under daily cycles and constant conditions. In support to its circadian nature, cycling is lost in bona fide clockless mutants. We propose this clock-controlled structural plasticity as a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells.

The axon-guidance roundabout gene alters the pace of the Drosophila circadian clock.

Great efforts have been directed to the dissection of the cell-autonomous circadian oscillator in Drosophila. However, less information is available regarding how this oscillator controls rhythmic rest-activity cycles. We have identified a viable allele of roundabout, robo(hy), where the period of locomotor activity is shortened. From its role in axon-pathfinding, we anticipated developmental defects in clock-relevant structures. However, robo(hy) produced minor defects in the architecture of the circuits essential for rhythmic behaviour. ROBO's presence within the circadian circuit strengthened the possibility of a novel role for ROBO at this postdevelopmental stage. Genetic interactions between pdf (01) and robo(hy) suggest that ROBO could alter the communication within different clusters of the circadian network, thus impinging on two basic properties, periodicity and/or rhythmicity. Early translocation of PERIOD to the nucleus in robo(hy) pacemaker cells indicated that shortened activity rhythms were derived from alterations in the molecular oscillator. Herein we present a mutation affecting clock function associated with a molecule involved in circuit assembly and maintenance.

Impaired clock output by altered connectivity in the circadian network.

Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.

Neuronal death in Drosophila triggered by GAL4 accumulation.

The GAL4/UAS system has been extensively employed in Drosophila to control gene expression in defined spatial patterns. More recently this system has been successfully applied to express genes involved in neurodegeneration to model various diseases in the fruit fly. We used transgenic lines expressing different levels of GAL4 in a particular subset of neurons involved in the control of rhythmic behaviour, so that its impact on neuronal physiology would result in altered locomotor activity, which could be readily assessed. We observed a striking correlation between gal4 dosage and behavioural defects associated with apoptotic neuronal loss in the specific GAL4-expressing neurons. Increased gal4 dosage correlated with accumulation of insoluble GAL4, suggesting that the cascade of events leading to apoptosis might be triggered by protein deposits of either GAL4 or protein intermediates. Behavioural defects were rescued by expression of hsp70, a classic chaperone that also interferes with cell death pathways. In agreement with the latter, the viral caspase inhibitor p35 also rescued GAL4-induced behavioural defects. Our observations demonstrate the intrinsic effects of GAL4 deregulation on neuronal viability and suggest that an excess of GAL4 might enhance neuronal deficits observed in models of neurodegeneration.

Enfoques genómicos para el estudio del control del comportamiento en Drosophila

El objetivo específico de este proyecto es la caracterización del rol del canal de potasio s/owpokey su proteína regulatoria (slob) en el control rítmico del comportamiento.Como primera medida se confirmaron los resultados obtenidos en los experimentos de chips para dos genes potencialmente interesantes: slob (northern blot) y slo (RT-PCR). En el caso del canal fue posible además evaluar las oscilaciones en los niveles de proteína ya que contamos con un anticuerpo específico. Posteriormente se evaluó a nivel comportamental dos mutantes nulas para la función del canal: sloly slo4. Ante nuestra sorpresa ambas resultaron significativamente mas arrítmicas que su control genético correspondiente (Cantón S, CS). Como se mencionó en el informe anterior, s/c4 es el resultado de una inversión que afecta un número importante de genes (13 conocidos y varias decenas más de genes predichos por algoritmos, o *CG’s) Ins

Enfoques genómicos para el estudio del control del comportamiento en Drosophila

El objetivo específico de este proyecto es la caracterización del rol del canal de potasio s/owpokey su proteína regulatoria (slob) en el control rítmico del comportamiento.Como primera medida se confirmaron los resultados obtenidos en los experimentos de chips para dos genes potencialmente interesantes: slob (northern blot) y slo (RT-PCR). En el caso del canal fue posible además evaluar las oscilaciones en los niveles de proteína ya que contamos con un anticuerpo específico. Posteriormente se evaluó a nivel comportamental dos mutantes nulas para la función del canal: sloly slo4. Ante nuestra sorpresa ambas resultaron significativamente mas arrítmicas que su control genético correspondiente (Cantón S, CS). Como se mencionó en el informe anterior, s/c4 es el resultado de una inversión que afecta un número importante de genes (13 conocidos y varias decenas más de genes predichos por algoritmos, o *CG’s) Ins

Enfoques genómicos para el estudio del control del comportamiento en Drosophila

El objetivo específico de este proyecto es la caracterización del rol del canal de potasio s/owpokey su proteína regulatoria (slob) en el control rítmico del comportamiento.Como primera medida se confirmaron los resultados obtenidos en los experimentos de chips para dos genes potencialmente interesantes: slob (northern blot) y slo (RT-PCR). En el caso del canal fue posible además evaluar las oscilaciones en los niveles de proteína ya que contamos con un anticuerpo específico. Posteriormente se evaluó a nivel comportamental dos mutantes nulas para la función del canal: sloly slo4. Ante nuestra sorpresa ambas resultaron significativamente mas arrítmicas que su control genético correspondiente (Cantón S, CS). Como se mencionó en el informe anterior, s/c4 es el resultado de una inversión que afecta un número importante de genes (13 conocidos y varias decenas más de genes predichos por algoritmos, o *CGs) Ins