TBI and Tau Loss of Function Both Affect Naïve Ethanol Sensitivity in Drosophila.

Traumatic brain injury (TBI) is associated with alcohol abuse and higher ethanol sensitivity later in life. Currently, it is poorly understood how ethanol sensitivity changes with time after TBI and whether there are sex-dependent differences in the relationship between TBI and ethanol sensitivity. This study uses the fruit fly Drosophila melanogaster to investigate how TBI affects alcohol sensitivity and whether the effects are sex-specific. Our results indicate that flies have a significantly higher sensitivity to the intoxicating levels of ethanol during the acute phase post-TBI, regardless of sex. The increased ethanol sensitivity decreases as time progresses; however, females take longer than males to recover from the heightened ethanol sensitivity. Dietary restriction does not improve the negative effects of alcohol post-TBI. We found that tau mutant flies exhibit a similar ethanol sensitivity to TBI flies. However, TBI increased the ethanol sensitivity of dtauKO mutants, suggesting that TBI and dtau loss of function have additive effects on ethanol sensitivity.

Mutants of the white ABCG Transporter in Drosophila melanogaster Have Deficient Olfactory Learning and Cholesterol Homeostasis.

Drosophila's white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.

Normal Ethanol Sensitivity and Rapid Tolerance Require the G Protein Receptor Kinase 2 in Ellipsoid Body Neurons in Drosophila.

BACKGROUND: G protein signaling pathways are key neuromodulatory mechanisms for behaviors and neurological functions that affect the impact of ethanol (EtOH) on locomotion, arousal, and synaptic plasticity. Here, we report a novel role for the Drosophila G protein-coupled receptor kinase 2 (GPRK2) as a member of the GRK4/5/6 subfamily in modulating EtOH-induced behaviors. METHODS: We studied the requirement of Drosophila Gprk2 for naïve sensitivity to EtOH sedation and ability of the fly to develop rapid tolerance after a single exposure to EtOH, using the loss of righting reflex (LORR) and fly group activity monitor (FlyGrAM) assays. RESULTS: Loss-of-function Gprk2 mutants demonstrate an increase in alcohol-induced hyperactivity, reduced sensitivity to the sedative effects of EtOH, and diminished rapid tolerance after a single intoxicating exposure. The requirement for Gprk2 in EtOH sedation and rapid tolerance maps to ellipsoid body neurons within the Drosophila brain, suggesting that wild-type Gprk2 is required for modulation of locomotion and alertness. However, even though Gprk2 loss of function leads to decreased and fragmented sleep, this change in the sleep state does not depend on Gprk2 expression in the ellipsoid body. CONCLUSION: Our work on GPRK2 has established a role for this GRK4/5/6 subfamily member in EtOH sensitivity and rapid tolerance.

Differentially timed extracellular signals synchronize pacemaker neuron clocks.

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. We also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.

PySmooth: a Python tool for the removal and correction of genotyping errors.

In genetic mapping studies involving many individuals, genome-wide markers such as single nucleotide polymorphisms (SNPs) can be detected using different methods. However, it comes with some errors. Some SNPs associated with diseases can be in regions encoding long noncoding RNAs (lncRNAs). Therefore, identifying the errors in genotype file and correcting them is crucial for accurate genetic mapping studies. We develop a Python tool called PySmooth, that offers an easy-to-use command line interface for the removal and correction of genotyping errors. PySmooth uses the approach of a previous tool called SMOOTH with some modifications. It inputs a genotype file, detects errors and corrects them. PySmooth provides additional features such as imputing missing data, better user-friendly usage, generates summary and visualization files, has flexible parameters, and handles more genotype codes. AVAILABILITY AND IMPLEMENTATION: PySmooth is available at https://github.com/lncRNAAddict/PySmooth .

Analysis of Positional Preference in Drosophila Using Multibeam Activity Monitors.

The positional preference of an animal can be very informative regarding the choices it makes about how to interact with its environment. The fruit fly Drosophila melanogaster has been used as a robust system for examining neurobiological mechanisms underlying behavior. Fruit fly positional preference can be gathered from TriKinetics Drosophila activity monitors (DAMs), which contain four infrared beams, allowing for tracking the position of individual flies along the length of a tube. Here, we describe a method for using DAM5Ms to examine food preference. Specifically, we show an example in which circadian changes in food preference are compared between different Drosophila species. More information about the evolution of behavior can be gathered by measuring feeding preference relative to time of day. Noni, fruit from Morinda citrifolia, contains octanoic acid, a chemical toxic to many species of Drosophila D. melanogaster and D. simulans, both food generalists, show high sensitivity to octanoic acid, whereas D. sechellia, a specialist, can tolerate high concentrations. When two different food substrates are provided at each end of a tube, food preference can be inferred at various times of the day, using the sleep and circadian analysis MATLAB program (SCAMP) to extract and analyze positional data from DAM5Ms. Data gathered from these analyses can be used to compare avoidance or attraction to nutrients, tastants, or odors between species and genotypes or after specific different treatments. Additionally, such data can be examined as a function of time of day.

Analysis of Sleep and Circadian Rhythms from Drosophila Activity-Monitoring Data Using SCAMP.

Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated and what biological functions it plays. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using beam break counts from Drosophila activity monitors (DAMs). The number of laboratories worldwide studying sleep in Drosophila has grown from only a few 20 years ago to hundreds today. The utility of these studies is limited by the quality of the metrics that can be extracted from the data. Many software options exist to help analyze DAM data; however, these are often expensive or have significant limitations. Therefore, we describe here a method for analyzing DAM-based data using the sleep and circadian analysis MATLAB program (SCAMP). This user-friendly software has an advantage of combining several analyses of both sleep and circadian rhythms in one package and produces graphical outputs as well as spreadsheets of the outputs for further statistical analysis. The version of SCAMP described here is also the first published software package that can analyze data from multibeam DAM5Ms, enabling determination of positional preference over time.

Ionic Liquid Coating-Driven Nanoparticle Delivery to the Brain: Applications for NeuroHIV.

Delivering cargo to the central nervous system (CNS) remains a pharmacological challenge. For infectious diseases such as HIV, the CNS acts as a latent reservoir that is inadequately managed by systemic antiretrovirals (ARTs). ARTs thus cannot eradicate HIV, and given CNS infection, patients experience neurological deficits collectively referred to as "neuroHIV". Herein, the development of bioinspired ionic liquid-coated nanoparticles (IL-NPs) for in situ hitchhiking on red blood cells (RBCs) is reported, which enables 48% brain delivery of intracarotid arterial- infused cargo. Moreover, IL choline trans-2-hexenoate (CA2HA 1:2) demonstrates preferential accumulation in parenchymal microglia over endothelial cells post-delivery. This study further demonstrates successful loading of abacavir (ABC), an ART that is challenging to encapsulate, into IL-NPs, and verifies retention of antiviral efficacy in vitro. IL-NPs are not cytotoxic to primary human peripheral blood mononuclear cells (PBMCs) and the CA2HA 1:2 coating itself confers notable anti-viremic capacity. In addition, in vitro cell culture assays show markedly increased uptake of IL-NPs into neural cells compared to bare PLGA nanoparticles. This work debuts bioinspired ionic liquids as promising nanoparticle coatings to assist CNS biodistribution and has the potential to revolutionize the delivery of cargos (i.e., drugs, viral vectors) through compartmental barriers such as the blood-brain-barrier (BBB).

The pre-synaptic Munc13-1 binds alcohol and modulates alcohol self-administration in Drosophila.

Munc13-1 is a pre-synaptic active-zone protein essential for neurotransmitter release and involved in pre-synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13-1 with EC50 s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13-1 C1 exclusively at Glu-582, which was identified by mass spectrometry. Mutation of Glu-582 to alanine, leucine, and histidine reduced the alcohol binding two- to five-fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild-type Munc13-1 compared with the mutants. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Dunc-13 in Drosophila melanogaster. The Dunc-13(P84200) /+ heterozygotes have 50% wild-type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. The present studies indicate that Munc13-1 C1 has binding site(s) for alcohols and Munc13-1 activity is sufficient to restore normal self-administration to Drosophila mutants deficient in Dunc-13 activity. The pre-synaptic Mun13-1 protein is a critical regulator of synaptic vesicle fusion and may be involved in processes that lead to ethanol abuse and addiction. We studied its interaction with alcohol and identified Glu-582 as a critical residue for ethanol binding. Munc13-1 can functionally complement the Dunc13 haploinsufficient ethanol self-administration phenotype in Drosophila melanogaster, indicating that this protein participates in alcohol-induced behavioral plasticity.

Carbon dioxide sensing modulates lifespan and physiology in Drosophila.

For nearly all life forms, perceptual systems provide access to a host of environmental cues, including the availability of food and mates as well as the presence of disease and predators. Presumably, individuals use this information to assess the current and future states of the environment and to enact appropriate developmental, behavioral, and regulatory decisions. Recent work using the nematode worm, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster, has established that aging is subject to modulation through neurosensory systems and that this regulation is evolutionarily conserved. To date, sensory manipulations shown to impact Drosophila aging have involved general loss of function or manipulation of complex stimuli. We therefore know little about the specific inputs, sensors, or associated neural circuits that affect these life and death decisions. We find that a specialized population of olfactory neurons that express receptor Gr63a (a component of the olfactory receptor for gaseous phase CO(2)) affects fly lifespan and physiology. Gr63a loss of function leads to extended lifespan, increased fat deposition, and enhanced resistance to some (but not all) environmental stresses. Furthermore, we find that the reduced lifespan that accompanies exposure to odors from live yeast is dependent on Gr63a. Together these data implicate a specific sensory cue (CO(2)) and its associated receptor as having the ability to modulate fly lifespan and alter organism stress response and physiology. Because Gr63a is expressed in a well-defined population of neurons, future work may now be directed at dissecting more complex neurosensory and neuroendocrine circuits that modulate aging in Drosophila.

G(o) activation is required for both appetitive and aversive memory acquisition in Drosophila.

Heterotrimeric G(o) is an abundant brain protein required for negatively reinforced short-term associative olfactory memory in Drosophila. G(o) is the only known substrate of the S1 subunit of pertussis toxin (PTX) in fly, and acute expression of PTX within the mushroom body neurons (MB) induces a reversible deficit in associative olfactory memory. We demonstrate here that the induction of PTX within the α/ß and γ lobe MB neurons leads to impaired memory acquisition without affecting memory stability. The induction of PTX within these MB neurons also leads to a significant defect in an optimized positively reinforced short-term memory paradigm; however, this PTX-induced learning deficit is noticeably less severe than found with the negatively reinforced paradigm. Both negatively and positively reinforced memory phenotypes are rescued by the constitutive expression of G(o)α transgenes bearing the Cys(351)Ile mutation. Since this mutation renders the G(o) molecule insensitive to PTX, the results isolate the effect of PTX on both forms of olfactory associative learning to the inhibition of the G(o) activation.

Drosophila genotypes can be predicted from their exploration locomotive trajectories using supervised machine learning.

This study employs supervised machine learning algorithms to test whether locomotive features during exploratory activity in open field arenas can serve as predictors for the genotype of fruit flies. Because of the nonlinearity in locomotive trajectories, traditional statistical methods that are used to compare exploratory activity between genotypes of fruit flies may not reveal all insights. 10-minute-long trajectories of four different genotypes of fruit flies in an open-field arena environment were captured. Turn angles and step size features extracted from the trajectories were used for training supervised learning models to predict the genotype of the fruit flies. Using the first five minute locomotive trajectories, an accuracy of 83% was achieved in differentiating wild-type flies from three other mutant genotypes. Using the final 5 min and the entire ten minute duration decreased the performance indicating that the most variations between the genotypes in their exploratory activity are exhibited in the first few minutes. Feature importance analysis revealed that turn angle is a better predictor than step size in predicting fruit fly genotype. Overall, this study demonstrates that features of trajectories can be used to predict the genotype of fruit flies through supervised machine learning methods.

Ionic Liquid Coating-Driven Nanoparticle Delivery to the Brain: Applications for NeuroHIV.

Delivering cargo to the central nervous system (CNS) remains a pharmacological challenge. For infectious diseases such as HIV, the CNS acts as a latent reservoir that is inadequately managed by systemic antiretrovirals (ARTs). ARTs thus cannot eradicate HIV, and given CNS infection, patients experience an array of neurological deficits that are collectively referred to as 'neuroHIV'. Herein we report the development of bioinspired ionic liquid-coated nanoparticles (IL-NPs) for in situ hitchhiking on red blood cells (RBCs), which enabled 48% delivery of intravenously infused cargo to the brain. Moreover, the ionic liquid (IL) choline trans-2-hexenoate (CA2HA 1:2) demonstrated preferential accumulation in parenchymal microglia over endothelial cells post-delivery. We further demonstrate the successful loading of abacavir (ABC), an ART that is challenging to encapsulate, into the IL-coated NPs and verify the retention of antiviral efficacy in vitro. IL-NPs were not cytotoxic to primary human peripheral blood mononuclear cells (PBMCs) and the CA2HA 1:2 coating conferred notable anti-viremic capacity on its own. In addition, in vitro cell culture assays showed markedly increased uptake of IL-coated nanoparticles into neuronal cells compared to bare nanoparticles. This work debuts bioinspired ionic liquids as promising nanoparticle coatings to assist CNS biodistribution and has the potential to revolutionize the delivery of cargos (i.e., drugs, viral vectors) through compartmental barriers such as the blood-brain-barrier (BBB), illustrated in the graphical abstract below.

Quantum origins of molecular recognition and olfaction in Drosophila.

The standard model for molecular recognition of an odorant is that receptor sites discriminate by molecular geometry as evidenced that two chiral molecules may smell very differently. However, recent studies of isotopically labeled olfactants indicate that there may be a molecular vibration-sensing component to olfactory reception, specifically in the spectral region around 2300 cm(-1). Here, we present a donor-bridge-acceptor model for olfaction which attempts to explain this effect. Our model, based upon accurate quantum chemical calculations of the olfactant (bridge) in its neutral and ionized states, posits that internal modes of the olfactant are excited impulsively during hole transfer from a donor to acceptor site on the receptor, specifically those modes that are resonant with the tunneling gap. By projecting the impulsive force onto the internal modes, we can determine which modes are excited at a given value of the donor-acceptor tunneling gap. Only those modes resonant with the tunneling gap and are impulsively excited will give a significant contribution to the inelastic transfer rate. Using acetophenone as a test case, our model and experiments on D. melanogaster suggest that isotopomers of a given olfactant give rise to different odorant qualities. These results support the notion that inelastic scattering effects may play a role in discriminating between isotopomers but that this is not a general spectroscopic effect.

Differential Expression of Presynaptic Munc13-1 and Munc13-2 in Mouse Hippocampus Following Ethanol Drinking.

The Munc13 family of proteins is critically involved in synaptic vesicle priming and release in glutamatergic neurons in the brain. Munc13-1 binds to alcohol and, in Drosophila, modulates sedation sensitivity and self-administration. We examined the effect of alcohol consumption on the expression of Munc13-1 and Munc13-2, NMDA receptor subunits GluN1, GluN2A and GluN2B in the hippocampus-derived HT22 cells, hippocampal primary neuron culture, and wild-type and Munc13-1+/- male mouse hippocampus after ethanol consumption (Drinking in the Dark (DID) paradigm). In HT22 cells, Munc13-1 was upregulated following 25 mM ethanol treatment for 24 h. In the primary neuronal culture, however, the expression of both Munc13-1 and Munc13-2 increased after ethanol exposure. While Munc13-1 was upregulated in the hippocampus, Munc13-2 was downregulated following DID. This differential effect was found in the CA1 subfield of the hippocampus. Although Munc13-1+/- mice had approximately 50% Munc13-1 expression compared to wild-type, it was nonetheless significantly increased following DID. Munc13-1 and Munc13-2 were expressed in vesicular glutamate transporter1 (VGLUT1) immunoreactive neurons in the hippocampus, but ethanol did not alter the expression of VGLUT1. The NMDA receptor subunits, GluN1, GluN2A and GluN2B were upregulated in the hippocampal primary culture and in the CA1. Ethanol exerts a differential effect on the expression of Munc13-1 and Munc13-2 in the CA1 in male mice. Our study also found that ethanol's effect on Munc13 expression is dependent on the experimental paradigm, and both Munc13-1 and Munc13-2 could contribute to the ethanol-induced augmentation of glutamatergic neurotransmission.

Cycling behavior and memory formation.

Circadian research has spent considerable effort in the determining clock output pathways, including identifying both physiological and behavioral processes that demonstrate significant time-of-day variation. Memory formation and consolidation represent notable processes shaped by endogenous circadian oscillators. To date, very few studies on memory mechanisms have considered potential confounding effects of time-of-day and the organism's innate activity cycles (e.g., nocturnal, diurnal, or crepuscular). The following studies highlight recent work describing this interactive role of circadian rhythms and memory formation, and were presented at a mini-symposium at the 2009 annual meeting of the Society for Neuroscience. The studies illustrate these time-of-day observations in a variety of behavioral paradigms and model organisms, including olfactory avoidance conditioning in Drosophila, long-term sensitization in Aplysia, active-avoidance conditioning in Zebrafish, and classical fear conditioning in rodents, suggesting that the circadian influence on memory behavior is highly conserved across species. Evidence also exists for a conserved mechanistic relationship between specific cycling molecules and memory formation, and the extent to which proper circadian cycling of these molecules is necessary for optimal cognitive performance. Studies describe the involvement of the core clock gene period, as well as vasoactive intestinal peptide, melatonin, and the cAMP/MAPK (cAMP/mitogen-activated protein kinase) cascade. Finally, studies in humans describe evidence for alterations in cognitive performance based on an interaction between sleep-wake homeostasis and the internal circadian clock. Conservation of a functional relationship between circadian rhythms with learning and memory formation across species provides a critical framework for future analysis of molecular mechanisms underlying complex behavior.

A role for the adult fat body in Drosophila male courtship behavior.

Mating behavior in Drosophila depends critically on the sexual identity of specific regions in the brain, but several studies have identified courtship genes that express products only outside the nervous system. Although these genes are each active in a variety of non-neuronal cell types, they are all prominently expressed in the adult fat body, suggesting an important role for this tissue in behavior. To test its role in male courtship, fat body was feminized using the highly specific Larval serum protein promoter. We report here that the specific feminization of this tissue strongly reduces the competence of males to perform courtship. This effect is limited to the fat body of sexually mature adults as the feminization of larval fat body that normally persists in young adults does not affect mating. We propose that feminization of fat body affects the synthesis of male-specific secreted circulating proteins that influence the central nervous system. In support of this idea, we demonstrate that Takeout, a protein known to influence mating, is present in the hemolymph of adult males but not females and acts as a secreted protein.

A theory for the arrangement of sensory organs in Drosophila.

We study the arrangements of recurved bristles on the anterior wing margin of wild-type and mutant Drosophila. The epidermal or neural fate of a proneural cell depends on the concentrations of proteins of the achaete-scute complex. At puparium formation, concentrations of proteins are nearly identical in all cells of the anterior wing and each cell has the potential for neural fate. In wild-type flies, the action of regulatory networks drives the initial state to one where a bristle grows out of every fifth cell. Recent experiments have shown that the frequency of recurved bristles can be made to change by adjusting the mean concentrations of the zinc-finger transcription factor Senseless and the micro-RNA miR-9a. Specifically, mutant flies with reduced levels of miR-9a exhibit ectopic bristles, and those with lower levels of both miR-9a and Senseless show regular organization of recurved bristles, but with a lower periodicity of 4. We argue that these characteristics can be explained assuming an underlying Turing-type bifurcation whereby a periodic pattern spontaneously emerges from a uniform background. However, bristle patterns occur in a discrete array of cells, and are not mediated by diffusion. We argue that intracellular actions of transmembrane proteins such as Delta and Notch can play a role of diffusion in destabilizing the homogeneous state. In contrast to diffusion, intercellular actions can be activating or inhibiting; further, there can be lateral cross-species interactions. We introduce a phenomenological model to study bristle arrangements and make several model-independent predictions that can be tested in experiments. In our theory, miRNA-9a is one of the components of the underlying network and has no special regulatory role. The loss of periodicity in its absence is due to the transfer of the system to a bistable state.

G(o) signaling is required for Drosophila associative learning.

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. Here we demonstrate that G(o) signaling is required for the formation of associative memory. In Drosophila melanogaster, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both gamma- and alpha/beta-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory.

Circadian modulation of short-term memory in Drosophila.

Endogenous biological clocks are widespread regulators of behavior and physiology, allowing for a more efficient allocation of efforts and resources over the course of a day. The extent that different processes are regulated by circadian oscillators, however, is not fully understood. We investigated the role of the circadian clock on short-term associative memory formation using a negatively reinforced olfactory-learning paradigm in Drosophila melanogaster. We found that memory formation was regulated in a circadian manner. The peak performance in short-term memory (STM) occurred during the early subjective night with a twofold performance amplitude after a single pairing of conditioned and unconditioned stimuli. This rhythm in memory is eliminated in both timeless and period mutants and is absent during constant light conditions. Circadian gating of sensory perception does not appear to underlie the rhythm in short-term memory as evidenced by the nonrhythmic shock avoidance and olfactory avoidance behaviors. Moreover, central brain oscillators appear to be responsible for the modulation as cryptochrome mutants, in which the antennal circadian oscillators are nonfunctional, demonstrate robust circadian rhythms in short-term memory. Together these data suggest that central, rather than peripheral, circadian oscillators modulate the formation of short-term associative memory and not the perception of the stimuli.