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Preference for spatial locations to maximize favorable outcomes and minimize aversive experiences helps animals survive and adapt to the changing environment. Both visual and non-visual cues play a critical role in spatial navigation and memory of a place supports and guides these strategies. Here we present the neural, genetic and behavioral processes involved in place memory formation using

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a high-throughput approach to study neural regulation of sleep and factors that affect this complex behavior.

Neural circuits involved in regulation of sleep play a critical role in sleep-wake transition and ability of an organism to engage in other behaviors critical for survival. The fruit fly, Drosophila melanogaster is a powerful system for the study of sleep and circuit mechanisms underlying sleep and co-regulation of sleep with other behaviors. InDrosophila, two neuropils in the central brain, mushroom body (MB) and central complex (CX) have been shown to influence sleep homeostasis and receive neuromodulator input critical to sleep-wake switch.Dopamine neurons (DANs) are the primary neuromodulator inputs to the MB but the mechanisms by which they regulate sleep- and wake-promoting neurons within MB are unknown. Here we investigate the role of subsets of DANs that signal wakefulness and project to wake-promoting compartments of the MB. We find that inhibition of specific subsets of PAM and PPL1 DANs projecting to the MB increase sleep in the presence of strong wake-inducing stimuli that reduces GABA transmission, although activity of these neurons is not directly modulated by GABA signaling. Of these subsets we find that DANs innervating the γ5 and β’2 MB compartments require both DopR1 and DopR2 receptors located in downstream Kenyon cells and mushroom body output neurons (MBONs). Further, we report that unlike the activity of wake-promoting MBONs and KCs, whose activity is modulated by sleep-need and PAM-DAN activity is independent of sleep-need. We have characterized a dopamine mediated sleep-circuit providing an inroad into understanding how common circuits within MB regulate sleep, wakefulness and behavioral arousal.

Troy D. Zars (1967–2018) was an American biologist. He studied the relationships between genes, neuronal circuits and behavior in the fruit fly Drosophila melanogaster. Zars co-pioneered the use of...

Balance between sleep, wakefulness and arousal is important for survival of organisms and species as a whole. While, the benefits of sleep both in terms of quantity and quality is widely recognized across species, sleep has a cost for organismal survival and reproduction. Here we focus on how sleep duration, sleep depth and sleep pressure affect the ability of animals to engage in courtship and egg-laying behaviors critical for reproductive success. Using isogenic lines from the Drosophila Genetic Reference Panel with variable sleep phenotypes we investigated the relationship between sleep and reproductive behaviors, courtship and oviposition. We found that three out of five lines with decreased sleep and increased arousal phenotypes, showed increased courtship and decreased latency to court as compared to normal and long sleeping lines. However, the male courtship phenotype is dependent on context and genotype as some but not all long sleeping-low courting lines elevate their courtship in the presence of short sleeping-high courting flies. We also find that unlike courtship, sleep phenotypes were less variable and minimally susceptible to social experience. In addition to male courtship, we also investigated egg-laying phenotype, a readout of female reproductive output and find oviposition to be less sensitive to sleep length and parameters that are indicative of switch between sleep and wake states. Taken together our extensive behavioral analysis here shows complex bidirectional interactions between genotype and environment and add to the growing evidence linking sleep duration and sleep-wake switch parameters to behavioral decision making critical to reproductive output.

This review serves as an introduction to a Special Issue of Comparative Biochemistry and Physiology, focused on using non-human models to study biomedical physiology. The concept of a model differs across disciplines. For example, several models are used primarily to gain an understanding of specific human pathologies and disease states, whereas other models may be focused on gaining insight into developmental or evolutionary mechanisms. It is often the case that animals initially used to gain knowledge of some unique biochemical or physiological process finds foothold in the biomedical community and becomes an established model. The choice of a particular model for biomedical research is an ongoing process and model validation must keep pace with existing and emerging technologies. While the importance of non-mammalian models, such as Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Xenopus laevis, is well known, we also seek to bring attention to emerging alternative models of both invertebrates and vertebrates, which are less established but of interest to the comparative biochemistry and physiology community.

Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. Here we studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. We show that an increased need for sleep inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fru M and dsx, but does not affect female sexual behavior. Further, we delineate a sex-specific neuronal circuit wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fru M-positive sleep-controlling DN1 neurons. In addition, we find that FRUM regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated., Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. Here the authors identify the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 255, 2021, 110593, https://doi.org/10.1016/j.cbpb.2021.110593. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a high-throughput approach to study neural regulation of sleep and factors that affect this complex behavior.

The Drosophila mushroom body (MB) is a key associative memory center that has also been implicated in the control of sleep. However, the identity of MB neurons underlying homeostatic sleep regulation, as well as the types of sleep signals generated by specific classes of MB neurons, has remained poorly understood. We recently identified two MB output neuron (MBON) classes whose axons convey sleep control signals from the MB to converge in the same downstream target region: a cholinergic sleep-promoting MBON class and a glutamatergic wake-promoting MBON class. Here, we deploy a combination of neurogenetic, behavioral, and physiological approaches to identify and mechanistically dissect sleep-controlling circuits of the MB. Our studies reveal the existence of two segregated excitatory synaptic microcircuits that propagate homeostatic sleep information from different populations of intrinsic MB "Kenyon cells" (KCs) to specific sleep-regulating MBONs: sleep-promoting KCs increase sleep by preferentially activating the cholinergic MBONs, while wake-promoting KCs decrease sleep by preferentially activating the glutamatergic MBONs. Importantly, activity of the sleep-promoting MB microcircuit is increased by sleep deprivation and is necessary for homeostatic rebound sleep (i.e., the increased sleep that occurs after, and in compensation for, sleep lost during deprivation). These studies reveal for the first time specific functional connections between subsets of KCs and particular MBONs and establish the identity of synaptic microcircuits underlying transmission of homeostatic sleep signals in the MB.

A key element of laboratory courses introducing students to neuroscience includes behavioral exercises. Associative learning experiments often conducted in research laboratories are difficult to perform and time consuming. Commonly, these experiments cannot be performed without extensive instrumentation or animal care facilities. Here, we describe three distinct laboratory modules that build on simple chemosensory and memory assays in Drosophila larvae. Additionally, we describe open-ended research projects using these assays that can be developed into semester long independent research experiences. Given that Drosophila is a genetic model organism, these simple behavioral assays can be used to generate multiple hypothesis driven projects aimed at identifying a gene or class of neurons involved in appetitive and aversive learning. These lab modules are ideally suited for undergraduates at all levels to experience and can be incorporated in a lower/upper level neuroscience course or as a high school outreach exercise. Further, these modules enable students to collect their own data sets, work in groups in collating large data sets, performing statistical comparisons, and presenting results in the form of short research papers or traditional laboratory reports that include a short literature review.

Feedback mechanisms in operant learning are critical for animals to increase reward or reduce punishment. However, not all conditions have a behavior that can readily resolve an event. Animals must then try out different behaviors to better their situation through outcome learning. This form of learning allows for novel solutions and with positive experience can lead to unexpected behavioral routines. Learned helplessness, as a type of outcome learning, manifests in part as increases in escape latency in the face of repeated unpredicted shocks. Little is known about the mechanisms of outcome learning. When fruit fly Drosophila melanogaster are exposed to unpredicted high temperatures in a place learning paradigm, flies both increase escape latencies and have a higher memory when given control of a place/temperature contingency. Here we describe discrete serotonin neuronal circuits that mediate aversive reinforcement, escape latencies, and memory levels after place learning in the presence and absence of unexpected aversive events. The results show that two features of learned helplessness depend on the same modulatory system as aversive reinforcement. Moreover, changes in aversive reinforcement and escape latency depend on local neural circuit modulation, while memory enhancement requires larger modulation of multiple behavioral control circuits.

The mechanisms by which clock neurons in the Drosophila brain confer an ∼24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.

The biogenic amines dopamine, octopamine, and serotonin are critical in establishing normal memories. A common view for the amines in insect memory performance has emerged in which dopamine and octopamine are largely responsible for aversive and appetitive memories. Examination of the function of serotonin begins to challenge the notion of one amine type per memory because altering serotonin function also reduces aversive olfactory memory and place memory levels. Could the function of serotonin be restricted to the aversive domain, suggesting a more specific dopamine/serotonin system interaction? The function of the serotonergic system in appetitive olfactory memory was examined. By targeting the tetanus toxin light chain (TNT) and the human inwardly rectifying potassium channel (Kir2.1) to the serotonin neurons with two different GAL4 driver combinations, the serotonergic system was inhibited. Additional use of the GAL80(ts1) system to control expression of transgenes to the adult stage of the life cycle addressed a potential developmental role of serotonin in appetitive memory. Reduction in appetitive olfactory memory performance in flies with these transgenic manipulations, without altering control behaviors, showed that the serotonergic system is also required for normal appetitive memory. Thus, serotonin appears to have a more general role in Drosophila memory, and implies an interaction with both the dopaminergic and octopaminergic systems.

SUMMARY Animals receive rewards and punishments in different patterns. Sometimes stimuli or behaviors can become predictors of future good or bad events. Through learning, experienced animals can then avoid new but similar bad situations, or actively seek those conditions that give rise to good results. Not all good or bad events, however, can be accurately predicted. Interestingly, unpredicted exposure to presumed rewards or punishments can inhibit or enhance later learning, thus linking the two types of experiences. In Drosophila, place memories can be readily formed; indeed, memory was enhanced by exposing flies to high temperatures that are unpaired from place or behavioral contingencies. Whether it is the exposure to high temperatures per se or the lack of prediction about the exposure that is crucial for memory enhancement is unknown. Through yoking experiments, we show that the uncertainty about exposure to high temperatures positively biases later place memory. However, the unpredicted exposures to high temperature do not alter thermosensitivity. Thus, the uncertainty bias does not alter thermosensory processes. An unidentified system is proposed to buffer the high-temperature reinforcement information to influence place learning when accurate predictions can be identified.

Biogenic amines, such as serotonin and dopamine, can be important in reinforcing associative learning. This function is evident as changes in memory performance with manipulation of either of these signals. In the insects, evidence begins to argue for a common role of dopamine in negatively reinforced memory. In contrast, the role of the serotonergic system in reinforcing insect associative learning is either unclear or controversial. We investigated the role of both of these signals in operant place learning in Drosophila . By genetically altering serotonin and dopamine levels, manipulating the neurons that make serotonin and dopamine, and pharmacological treatments we provide clear evidence that serotonin, but not dopamine, is necessary for place memory. Thus, serotonin can be critical for memory formation in an insect, and dopamine is not a universal negatively reinforcing signal.

The ad hoc genetic correlation between ethanol sensitivity and learning mechanisms in Drosophila could overemphasize a common process supporting both behaviors. To challenge directly the hypothesis that these mechanisms are singular, we examined the learning phenotypes of 10 new strains. Five of these have increased ethanol sensitivity, and the other 5 do not. We tested place and olfactory memory in each of these lines and found two new learning mutations. In one case, altering the tribbles gene, flies have a significantly reduced place memory, elevated olfactory memory, and normal ethanol response. In the second case, mutation of a gene we name ethanol sensitive with low memory (elm), place memory was not altered, olfactory memory was sharply reduced, and sensitivity to ethanol was increased. In sum, however, we found no overall correlation between ethanol sensitivity and place memory in the 10 lines tested. Furthermore, there was a weak but nonsignificant correlation between ethanol sensitivity and olfactory learning. Thus, mutations that alter learning and sensitivity to ethanol can occur independently of each other and this implies that the set of genes important for both ethanol sensitivity and learning is likely a subset of the genes important for either process.

Apparently unpaired exposure to appetitive or aversive stimuli can suppress or enhance later associative learning. While the suppressive effect has been found in both vertebrate and invertebrate animals, it is not clear if the enhancing effect is restricted to the vertebrates. Additionally, whether Drosophila associative learning can be influenced in either direction is open. To address these questions, we examined the effects of pre-exposing flies to a high temperature negative reinforcer in the heat-box place-learning paradigm. We found that pre-exposing flies to an unavoidable high temperature enhanced later associative conditioning that uses mild increases in temperature. This enhancement lasts at least 20 min, does not depend on changes in the straightforward avoidance behavior of a high temperature source, and is independent of the antennal thermosensor. We thus provide an example of enhanced associative learning after unpaired exposure to a typical reinforcer in an invertebrate animal, suggesting the conservation of this component of learning.

Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli are sparsely represented by ∼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection. DOI: http://dx.doi.org/10.7554/eLife.04580.001, eLife digest An animal's survival depends on its ability to respond appropriately to its environment, approaching stimuli that signal rewards and avoiding any that warn of potential threats. In fruit flies, this behavior requires activity in a region of the brain called the mushroom body, which processes sensory information and uses that information to influence responses to stimuli. Aso et al. recently mapped the mushroom body of the fruit fly in its entirety. This work showed, among other things, that the mushroom body contained 21 different types of output neurons. Building on this work, Aso et al. have started to work out how this circuitry enables flies to learn to associate a stimulus, such as an odor, with an outcome, such as the presence of food. Two complementary techniques—the use of molecular genetics to block neuronal activity, and the use of light to activate neurons (a technique called optogenetics)—were employed to study the roles performed by the output neurons in the mushroom body. Results revealed that distinct groups of output cells must be activated for flies to avoid—as opposed to approach—odors. Moreover, the same output neurons are used to avoid both odors and colors that have been associated with punishment. Together, these results indicate that the output cells do not encode the identity of stimuli: rather, they signal whether a stimulus should be approached or avoided. The output cells also regulate the amount of sleep taken by the fly, which is consistent with the mushroom body having a broader role in regulating the fly's internal state. The results of these experiments—combined with new knowledge about the detailed structure of the mushroom body—lay the foundations for new studies that explore associative learning at the level of individual circuits and their component cells. Given that the organization of the mushroom body has much in common with that of the mammalian brain, these studies should provide insights into the fundamental principles that underpin learning and memory in other species, including humans. DOI: http://dx.doi.org/10.7554/eLife.04580.002

The biogenic amines play a critical role in establishing memories. In the insects, octopamine, dopamine, and serotonin have key functions in memory formation. For Drosophila, octopamine is necessary and sufficient for appetitive olfactory memory formation. Whether octopamine plays a general role in reinforcing memories in the fly is not known. Place learning in the heat-box associates high temperatures with one part of a narrow chamber, and a cool, strongly preferred temperature with the other half of the chamber. The cool-temperature-associated chamber half could provide a rewarding stimulus to a fly, and thus a place memory is composed of an aversive and rewarded memory component. The role of octopamine in place memory was thus tested. Using a mutation in the tyramine beta hydroxylase (TbetaH[M18]) and blocking of evoked synaptic transmission in the octopamine (and tyramine) neurons labeled with a tyramine decarboxylase-2 (TDC2) gene regulatory elements we found that reinforcement of place memories is independent of normal octopamine signaling. Thus, reinforcing mechanisms in Drosophila have specialized systems in the formation of specific memory types.