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.

Sleep drive reconfigures wake-promoting clock circuitry to regulate adaptive behavior.

Circadian rhythms help animals synchronize motivated behaviors to match environmental demands. Recent evidence indicates that clock neurons influence the timing of behavior by differentially altering the activity of a distributed network of downstream neurons. Downstream circuits can be remodeled by Hebbian plasticity, synaptic scaling, and, under some circumstances, activity-dependent addition of cell surface receptors; the role of this receptor respecification phenomena is not well studied. We demonstrate that high sleep pressure quickly reprograms the wake-promoting large ventrolateral clock neurons to express the pigment dispersing factor receptor (PDFR). The addition of this signaling input into the circuit is associated with increased waking and early mating success. The respecification of PDFR in both young and adult large ventrolateral neurons requires 2 dopamine (DA) receptors and activation of the transcriptional regulator nejire (cAMP response element-binding protein [CREBBP]). These data identify receptor respecification as an important mechanism to sculpt circuit function to match sleep levels with demand.

Temporally and spatially partitioned neuropeptide release from individual clock neurons.

Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Here, imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca2+ data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca2+ influx, while somatic neuropeptide release is triggered by the biochemical signal IP3 Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron-dependent behaviors. Thus, compartment-specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction.

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-ß (PLCß) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Imaging Neuropeptide Release from Drosophila Clock and Motor Neurons.

Electrophysiological studies of synaptic function do not robustly report release of neuropeptides and neurotrophins. These limitations have been overcome with the presynaptic expression of optical release reporters based on green fluorescent protein and fluorogen-activating protein. Here we describe how to image neuropeptide release in Drosophila at the neuromuscular junction and in the adult brain.

GFP and FAP imaging of neuropeptide release in Drosophila.

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca2+ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP.

Circadian Vesicle Capture Prepares Clock Neuron Synapses for Daily Phase-Delayed Neuropeptide Release.

Drosophila sLNv clock neurons release the neuropeptide PDF to control circadian rhythms. Strikingly, PDF content in sLNv terminals is rhythmic with a peak in the morning hours prior to the onset of activity-dependent release. Because synaptic PDF accumulation, rather than synaptic release, aligns with the late-night elevations in both sLNv neuron excitability and Ca2+, we explored the dependence of presynaptic neuropeptide accumulation on neuropeptide vesicle transport, electrical activity and the circadian clock. Live imaging reveals that anterograde axonal transport is constant throughout the day and capture of circulating neuropeptide vesicles rhythmically boosts presynaptic neuropeptide content hours prior to release. The late-night surge in vesicle capture, like release, requires electrical activity and results in a large releasable pool of presynaptic vesicles to support the later burst of neuropeptide release. The circadian clock is also required suggesting that it controls the switch from vesicle capture to exocytosis, which are normally coupled activity-dependent processes. This toggling of activity transduction maximizes rhythmic synaptic neuropeptide release needed for robust circadian behavior and resolves the previously puzzling delay in timing of synaptic neuropeptide release relative to changes in sLNv clock neuron physiology.

Peptide-induced modulation of synaptic transmission and escape response in Drosophila requires two G-protein-coupled receptors.

Neuropeptides are found in both mammals and invertebrates and can modulate neural function through activation of G-protein-coupled receptors (GPCRS). The precise mechanisms by which many of these GPCRs modulate specific signaling cascades to regulate neural function are not well defined. We used Drosophila melanogaster as a model to examine both the cellular and behavioral effects of DPKQDFMRFamide, the most abundant peptide encoded by the dFMRF gene. We show that DPKQDFMRFamide enhanced synaptic transmission through activation of two G-protein-coupled receptors, Fmrf Receptor (FR) and Dromyosupressin Receptor-2 (DmsR-2). The peptide increased both the presynaptic Ca(2+) response and the quantal content of released transmitter. Peptide-induced modulation of synaptic function could be abrogated by depleting intracellular Ca(2+) stores or by interfering with Ca(2+) release from the endoplasmic reticulum through disruption of either the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor. The peptide also altered behavior. Exogenous DPKQDFMRFamide enhanced fictive locomotion; this required both the FR and DmsR-2. Likewise, both receptors were required for an escape response to intense light exposure. Thus, coincident detection of a peptide by two GPCRs modulates synaptic function through effects of Ca(2+)-induced Ca(2+) release, and we hypothesize that these mechanisms are involved in behavioral responses to environmental stress.

Equilibrative nucleoside transporter 2 regulates associative learning and synaptic function in Drosophila.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) in Drosophila. Null mutants of ent2 are lethal during late larval/early pupal stages, indicating that ent2 is essential for normal development. Hypomorphic mutant alleles of ent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock down ent2 expression in specific regions of the CNS and show that ent2 is required in the alpha/beta lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated in ent2 mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR(1)) into the ent2 mutant background. The results of the present study provide the first evidence of a role for ent2 function in Drosophila and suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

Presynaptic ryanodine receptor-activated calmodulin kinase II increases vesicle mobility and potentiates neuropeptide release.

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.

Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide.

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.

The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals.

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+ levels at rest and during stimulation.

Heat shock-mediated thermoprotection of larval locomotion compromised by ubiquitous overexpression of Hsp70 in Drosophila melanogaster.

Maintaining the competence of locomotor circuitry under stressful conditions can benefit organisms by enabling locomotion to more tolerable microhabitats. We show that prior heat shock protects locomotion and the locomotor central pattern generator of larval Drosophila against subsequent hyperthermic stress. We combined molecular genetic, electrophysiological, and behavioral techniques to investigate heat shock-mediated thermoprotection. Prior heat shock increased the distance traveled by larvae during hyperthermia before failure. The frequency of the rhythm of peristaltic locomotor contractions and the velocity of locomotion were both less thermosensitive after heat shock and were less susceptible to failure at high temperatures. Rhythmic coordinated motor patterns, recorded intracellularly as excitatory junction potentials in body wall muscles of dissected preparations, were centrally generated because patterns could still be generated in the absence of sensory feedback (sensory function disrupted with shibire). Prior heat shock protected central circuit operation during hyperthermic stress by increasing the temperature at which it failed. Overexpression of Hsp70 after a heat shock using transgenic flies (traII) did not enhance thermoprotection, as expected, but had deleterious effects on parameters of behavior.

Stress-induced thermoprotection of neuromuscular transmission.

Environmental stresses such as high temperature or low levels of oxygen can lead to structural destabilization of cells, disruption of cellular processes, and, in extreme cases, death. Previous experience of sub-lethal stress can lead to protection during a subsequent stress that may otherwise have been lethal. Synapses are particularly vulnerable to extreme environmental conditions and failure of function at this level may be the primary cause of organismal death. Prior heat shock induces enhanced thermotolerance at neuromuscular junctions in the locust extensor tibiae muscle and in abdominal muscles of larval Drosophila. Synaptic thermoprotection is associated with an increase in short-term plasticity at these synapses. Prior anoxic coma in locusts induces synaptic thermotolerance suggesting that the same protective pathways are activated. It is well established that diverse forms of stress induce the upregulation of cellular chaperones (heat shock proteins; HSPs) that mediate acquired protection. The mechanisms underlying HSP-mediated synaptic protection are currently unknown but evidence is accumulating that stabilization of the cytoskeleton may play an important role.

A role for the cytoskeleton in heat-shock-mediated thermoprotection of locust neuromuscular junctions.

A prior hyperthermic stress (heat shock) can induce thermoprotection of neuromuscular transmission in Locusta migratoria extensor tibiae muscle measured 4 h after the onset of the heat shock. It is not clear what effect an acute hyperthermic stress may have on the nervous system's ability to tolerate thermal stress, that is, before increased expression of heat-shock proteins. We found that over consecutive thermal stress tests, failure temperature was not altered in either heat-shock or control animals. This suggests that protective mechanisms are not established in the short term (within one hour). Various members of the heat-shock protein family interact with elements of the cytoskeleton. We found that preexposure of the preparation to cytoskeletal stabilizing drugs induced thermoprotection, while preexposure to cytoskeletal disrupting drugs disrupted the ability to confer and maintain thermoprotection. We conclude that thermoprotection relies on a stable cytoskeleton and suggest that members of the heat shock protein family are involved.