Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naïve Drosophila. Thirst also permits flies to learn olfactory cues paired with water reward. Water learning requires water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body γ lobe. These water learning neurons are different from those that are critical to convey the reinforcing effects of sugar. Naïve water-seeking behavior in thirsty flies does not require water taste but relies on another subset of water-responsive dopaminergic neurons that target the mushroom body β′ lobe. Furthermore, these naïve water-approach neurons are not required for learned water-seeking. Our results therefore demonstrate that naïve and learned water-seeking, and water learning, utilize separable neural circuitry in the brain of thirsty flies.
During olfactory learning in fruit flies, dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. Here we identify a class of downstream glutamatergic mushroom body output neurons (MBONs) called M4/6, or MBON-β2β′2a, MBON-β′2mp, and MBON-γ5β′2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the β, β′, and γ lobes. This anatomy and their odor tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bidirectionally alters the relative odor-drive of M4β′ neurons (MBON-β′2mp). Direct block of M4/6 neurons in naive flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into approach, while optogenetically activating these neurons induces avoidance behavior. We therefore propose that drive to the M4/6 neurons reflects odor-directed behavioral choice.
•Glutamatergic mushroom body output neurons are required for memory expression•Training bidirectionally alters relative odor drive to output neurons•Blocking glutamatergic mushroom body output neurons mimics appetitive conditioning•Optogenetic activation drives avoidance behavior
Fruit fly olfactory memory involves mushroom body plasticity. Owald et al. identified glutamatergic mushroom body output neurons that are critical for memory expression. Conditioning bidirectionally alters odor drive to these outputs. Blocking them mimics appetitive conditioning, whereas activation induces avoidance behavior.
Dopaminergic neurons provide reward learning signals in mammals and insects [1–4]. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars . Here, we tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. We found that dopaminergic neurons expressing the OAMB octopamine receptor  specifically convey the short-term reinforcing effects of sweet taste . These dopaminergic neurons project to the β′2 and γ4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory requires different dopaminergic neurons that project to the γ5b regions, and it can be artificially reinforced by those projecting to the β lobe and adjacent α1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occur in satiated flies, formation and expression of artificial long-term memory require flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population.
•Sweet taste and nutrient value recruit different reinforcing dopaminergic neurons•Sweetness and nutrient value separately reinforce short- and long-term memories•Reinforcement of short-term memory is not dependent on the state of hunger•Acquisition and retrieval of long-term memory are hunger state dependent
A small number of dopaminergic neurons in the fly brain are crucial for appetitive memory reinforcement. Huetteroth et al. further subdivide these rewarding neurons into those required for sweet taste reinforcement of short-term memory, nutrient-dependent long-term memory, and others that can artificially reinforce food-independent long-term memory.
Dopaminergic neurons provide value signals in mammals and insects [1–3]. During Drosophila olfactory learning, distinct subsets of dopaminergic neurons appear to assign either positive or negative value to odor representations in mushroom body neurons [4–9]. However, it is not known how flies evaluate substances that have mixed valence. Here we show that flies form short-lived aversive olfactory memories when trained with odors and sugars that are contaminated with the common insect repellent DEET. This DEET-aversive learning required the MB-MP1 dopaminergic neurons that are also required for shock learning . Moreover, differential conditioning with DEET versus shock suggests that formation of these distinct aversive olfactory memories relies on a common negatively reinforcing dopaminergic mechanism. Surprisingly, as time passed after training, the behavior of DEET-sugar-trained flies reversed from conditioned odor avoidance into odor approach. In addition, flies that were compromised for reward learning exhibited a more robust and longer-lived aversive-DEET memory. These data demonstrate that flies independently process the DEET and sugar components to form parallel aversive and appetitive olfactory memories, with distinct kinetics, that compete to guide learned behavior.
•Flies trained with unpalatable sugar learn the sweet-, nutrient, and bad-taste qualities•Distinct dopamine neurons reinforce the positive and negative memories in parallel•Early conditioned aversion switches to longer-lasting nutrient-memory-guided attraction•Flies remember individual qualities of a complex food source
Das et al. show that flies trained with unpalatable sugar learn both the nutritional and bad-taste qualities. Opposing memories are reinforced in parallel by distinct dopaminergic neurons. Conditioned behavior is initially aversive but soon switches to approach led by the longer-lasting nutrient-reinforced appetitive memory.
Reinforcement systems are believed to drive synaptic plasticity within neural circuits that store memories. Recent evidence from the fruit fly suggests that anatomically distinct dopaminergic neurons ultimately provide the key instructive signals for both appetitive and aversive learning. This dual role for dopamine overturns the previous model that octopamine signaled reward and dopamine punishment. More importantly, this anatomically segregated double role for dopamine in reward and aversion mirrors that emerging in mammals. Therefore, an antagonistic organization of distinct reinforcing dopaminegic neurons is a conserved feature of brains. It now seems crucial to understand how the dopaminergic neurons are controlled and what the released dopamine does to the underlying circuits to convey opposite valence.
Recent studies in mammals have documented neural expression and mobility of retrotransposons and suggested that neural genomes are diverse mosaics. Here we report transposition in memory-relevant neurons in the Drosophila brain. Cell-type specific gene expression profiling revealed transposon expression is more abundant in mushroom body (MB) αβ neurons than in neighboring MB neurons. The PIWI-RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αβ MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in brain. Lastly, paired-end deep sequencing identified over 200 de novo transposon insertions in αβ neurons, including events into memory-relevant loci. Our observations indicate that genomic heterogeneity is a conserved feature of the brain.
It is now almost forty years since the first description of learning in the fruit fly Drosophila melanogaster. Various incarnations of the classic mutagenesis approach envisaged in the early days have provided around one hundred learning defective mutant fly strains. Recent technological advances permit temporal control of neural function in the behaving fly. These approaches have radically changed experiments in the field and have provided a neural circuit perspective of memory formation, consolidation and retrieval. Combining neural perturbations with more classical mutant intervention allows investigators to interrogate the molecular and cellular processes of memory within the defined neural circuits. Here, we summarize some of the progress made in the last ten years that indicates a remarkable conservation of the neural mechanisms of memory formation between flies and mammals. We emphasize that considering an ethologically-relevant viewpoint might provide additional experimental power in studies of Drosophila memory.
In Drosophila, anatomically discrete dopamine neurons that innervate distinct zones of the mushroom body (MB) assign opposing valence to odors during olfactory learning. Subsets of MB neurons have temporally unique roles in memory processing, but valence-related organization has not been demonstrated. We functionally subdivided the αβ neurons, revealing a value-specific role for the ∼160 αβ core (αβc) neurons. Blocking neurotransmission from αβ surface (αβs) neurons revealed a requirement during retrieval of aversive and appetitive memory, whereas blocking αβc only impaired appetitive memory. The αβc were also required to express memory in a differential aversive paradigm demonstrating a role in relative valuation and approach behavior. Strikingly, both reinforcing dopamine neurons and efferent pathways differentially innervate αβc and αβs in the MB lobes. We propose that conditioned approach requires pooling synaptic outputs from across the αβ ensemble but only from the αβs for conditioned aversion.
•Differential representation of memory valence in Drosophila mushroom body neurons•αβ core neurons are specifically required for conditioned approach behavior•Relative aversive learning requires rewarding dopaminergic reinforcement•Distinct circuits drive learned aversion and approach
Perisse et al. demonstrate that discrete mushroom body neuron populations drive learned approach and avoidance behaviors in the fruit fly. Aversive and appetitive memories for the same odor are therefore represented in different neural ensembles.
Dopamine (DA) is synonymous with reward and motivation in mammals1,2. However, only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4. Instead octopamine (OA) has historically been considered the signal for reward in insects5–7. Here we show using temporal control of neural function in Drosophila that only short-term appetitive memory is reinforced by OA. Moreover, OA-dependent memory formation requires signaling through DA neurons. Part of the OA signal requires the α-adrenergic like OAMB receptor in an identified subset of mushroom body (MB)-targeted DA neurons. OA triggers an increase in intracellular calcium in these DA neurons and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking OA. Analysis of the β-adrenergic like Octβ2R receptor reveals that OA-dependent reinforcement also requires an interaction with DA neurons that control appetitive motivation. These data suggest that sweet taste engages a distributed OA signal that reinforces memory through discrete subsets of MB-targeted DA neurons. In addition, they reconcile prior findings with OA and DA and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged.
Dopamine (DA) is synonymous with reward and motivation in mammals1,2. However, only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4. Instead octopamine (OA) has historically been considered the signal for reward in insects5-7. Here we show using temporal control of neural function in Drosophila that only short-term appetitive memory is reinforced by OA. Moreover, OA-dependent memory formation requires signaling through DA neurons. Part of the OA signal requires the α-adrenergic like OAMB receptor in an identified subset of mushroom body (MB)-targeted DA neurons. OA triggers an increase in intracellular calcium in these DA neurons and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking OA. Analysis of the β-adrenergic like Octβ2R receptor reveals that OA-dependent reinforcement also requires an interaction with DA neurons that control appetitive motivation. These data suggest that sweet taste engages a distributed OA signal that reinforces memory through discrete subsets of MB-targeted DA neurons. In addition, they reconcile prior findings with OA and DA and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged.
Adrenergic signaling has important roles in synaptic plasticity and metaplasticity. However, the underlying mechanisms of these functions remain poorly understood. We investigated the role of octopamine, the invertebrate counterpart of adrenaline and noradrenaline, in synaptic and behavioral plasticity in Drosophila. We found that an increase in locomotor speed induced by food deprivation was accompanied by an activity- and octopamine-dependent extension of octopaminergic arbors and that the formation and maintenance of these arbors required electrical activity. Growth of octopaminergic arbors was controlled by a cAMP- and CREB-dependent positive-feedback mechanism that required Octpβ2R octopamine autoreceptors. Notably, this autoregulation was necessary for the locomotor response. In addition, octopamine neurons regulated the expansion of excitatory glutamatergic neuromuscular arbors through Octpβ2Rs on glutamatergic motor neurons. Our results provide a mechanism for global regulation of excitatory synapses, presumably to maintain synaptic and behavioral plasticity in a dynamic range.
Labile memory is thought to be held in the brain as persistent neural network activity [1–4]. However, it is not known how biologically relevant memory circuits are organized and operate. Labile and persistent appetitive memory in Drosophila requires output after training from the α′β′ subset of mushroom body (MB) neurons and from a pair of modulatory Dorsal Paired Medial (DPM) neurons [5–9]. DPM neurons innervate the entire MB lobe region and appear to be pre- and post-synaptic to the MB [7, 8], consistent with a recurrent network model. Here we identify a role after training for synaptic output from the GABAergic Anterior Paired Lateral (APL) neurons [10, 11]. Blocking synaptic output from APL neurons after training disrupts labile memory but does not affect long-term memory. APL neurons contact DPM neurons most densely in the α′β′ lobes although their processes are intertwined and contact throughout all the lobes. Furthermore, APL contacts MB neurons in the α′ lobe but makes little direct contact with those in the distal α lobe. We propose that APL neurons provide widespread inhibition to stabilize and maintain synaptic specificity of a labile memory trace in a recurrent DPM and MB α′β′ neuron circuit.
Taste is an early stage in food and drink selection for most animals [1, 2]. Detecting sweetness indicates the presence of sugar and possible caloric content. However, sweet taste can be an unreliable predictor of nutrient value because some sugars cannot be metabolized. In addition, discrete sugars are detected by the same sensory neurons in the mammalian  and insect gustatory systems [4, 5], making it difficult for animals to readily distinguish the identity of different sugars using taste alone [6–8]. Here we used an appetitive memory assay in Drosophila [9–11] to investigate the contribution of palatability and relative nutritional value of sugars to memory formation. We show that palatability and nutrient value both contribute to reinforcement of appetitive memory. Non-nutritious sugars formed less robust memory that could be augmented by supplementing with a tasteless but nutritious substance. Nutrient information is conveyed to the brain within minutes of training when it can be used to guide expression of a sugar-preference memory. Therefore flies can rapidly learn to discriminate between sugars using a post-ingestive reward evaluation system and they preferentially remember nutritious sugars.
Many molecular signals that represent hunger and satiety in the body have been identified, but relatively little is known about how these factors alter the nervous system to change behavior. Root et al. (2011) report that hunger modulates the sensitivity of specific olfactory sensory neurons in Drosophila and facilitates odor search behavior.
A goal of memory research is to understand how changing the weight of specific synapses in neural circuits in the brain leads to an appropriate learned behavioral response. Finding the relevant synapses should allow investigators to probe the underlying physiological and molecular operations that encode memories and permit their retrieval. In this review, I discuss recent work in Drosophila that implicates subsets of dopaminergic neurons in aversive reinforcement and appetitive motivation. The zonal architecture of these dopaminergic neurons may reveal the functional organization of aversive and appetitive memory in the mushroom bodies. Combinations of fly dopaminergic neurons might code negative and positive value consistent with a motivational systems role proposed in mammals.
Neural systems controlling the vital functions of sleep and feeding in mammals are tightly inter-connected: sleep deprivation promotes feeding, while starvation suppresses sleep. Here we show that starvation in Drosophila potently suppresses sleep suggesting that these two homeostatically regulated behaviors are also integrated in flies. The sleep suppressing effect of starvation is independent of the mushroom bodies, a previously identified sleep locus in the fly brain, and therefore is regulated by distinct neural circuitry. The circadian clock genes Clock (Clk) and cycle (cyc) are critical for proper sleep suppression during starvation. However, the sleep suppression is independent of light cues and of circadian rhythms because starved period mutants sleep like wild type flies. By selectively targeting subpopulations of Clk-expressing neurons we localize the observed sleep phenotype to the dorsally located circadian neurons. These findings show that Clk and cyc act during starvation to modulate the conflict of whether flies sleep or search for food.
Motivational states are important determinants of behavior. In fruit flies appetitive memory expression is constrained by satiety and promoted by hunger. Here we identify a neural mechanism that integrates the motivational state of hunger and memory. We show that stimulation of neurons that express Neuropeptide F (dNPF), an ortholog of mammalian NPY, mimicks food-deprivation and promotes memory performance in satiated flies. Robust appetitive memory performance requires the dNPF receptor in six dopaminergic neurons that innervate a distinct region of the mushroom bodies. Blocking these dopaminergic neurons releases memory performance in satiated flies whereas stimulation suppresses memory performance in hungry flies. Therefore dNPF and dopamine provide a motivational switch in the mushroom body that controls the output of appetitive memory.
The function of sleep is hotly contested. Two recent studies suggest that fly sleep may be required to rescale synapses in the brain.
Drosophila; learning; memory; behavior; plasticity
A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant receptor gene converge onto the same glomeruli in the brain (1–7). Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants (8–11). OSNs also exhibit odor-evoked responses with complex temporal dynamics (11), but the contribution of this activity to behavioral odor discrimination has received little attention (12). Here we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture, and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration indicating that they encode odorant identity. Therefore flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.
Although many animals use the Earth’s magnetic field for orientation and navigation1,2, the precise biophysical mechanisms underlying magnetic sensing have been elusive. One theoretical model proposes that geomagnetic fields are perceived by chemical reactions involving specialized photoreceptors3. But the specific photoreceptor involved in such magnetoreception has not been demonstrated conclusively in any animal. Here we show that the UV-A/blue light photoreceptor CRYPTOCHROME (CRY) is necessary for light-dependent magnetosensitive responses in Drosophila melanogaster. In a binary-choice behavioural assay for magnetosensitivity, wild-type flies exhibit significant naïve and trained responses to a magnetic field under full-spectrum light (~300–700 nm) but do not respond to the field when wavelengths in the CRY-sensitive, UV-A/blue part of the spectrum (<420 nm) are blocked. Remarkably, CRY-deficient cry0 and cryb flies do not show either naïve or trained responses to a magnet field under full-spectrum light. Moreover, CRY-dependent magnetosensitivity does not require a functioning circadian clock. Our work provides the first genetic evidence for a CRY-based magnetosensitive system in any animal.
In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, here we show that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body α′ β′ neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Last, experiments feeding and/or starving flies after training reveals a critical motivational drive that enables appetitive LTM retrieval.
memory formation; consolidation; olfactory; Drosophila; mechanisms; circuits
Drosophila mushroom bodies (MB) are bilaterally symmetric multi-lobed brain structures required for olfactory memory. Previous studies suggested that neurotransmission from MB neurons is only required for memory retrieval. Our unexpected observation that Dorsal Paired Medial (DPM) neurons, which project only to MB neurons, are required during memory storage but not for acquisition or retrieval, led us to revisit the role of MB neurons in memory processing. We show that neurotransmission from the α′β′ subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory but is dispensable during memory retrieval. In contrast neurotransmission from MB αβ neurons is only required for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in a MB α′β′ neuron-DPM neuron loop is required to stabilize memories formed in the MB αβ neurons.