Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning

Formation of normal olfactory memory requires the expression of the wild-type amnesiac gene in the dorsal paired medial (DPM) neurons. Imaging the activity in the processes of DPM neurons revealed that the neurons respond when the fly is stimulated with electric shock or with any odor that was tested. Pairing odor and electric-shock stimulation increases odor-evoked calcium signals and synaptic release from DPM neurons. These memory traces form in only one of the two branches of the DPM neuron process. Moreover, trace formation requires the expression of the wild-type amnesiac gene in the DPM neurons. The cellular memory traces first appear at 30 min after conditioning and persist for at least 1 hr, a time window during which DPM neuron synaptic transmission is required for normal memory. DPM neurons are therefore "odor generalists" and form a delayed, branch-specific, and amnesiac-dependent memory trace that may guide behavior after acquisition.     

Sequential use of mushroom body neuron subsets during drosophila odor memory processing

Drosophila mushroom bodies (MB) are bilaterally symmetric multilobed 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 during acquisition or retrieval, led us to revisit the role of MB neurons in memory processing. We show that neurotransmission from the alpha'beta' 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 alphabeta 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 an MB alpha'beta' neuron-DPM neuron loop is required to stabilize memories formed in the MB alphabeta neurons.     

Traces of Drosophila memory

Studies using functional cellular imaging of living flies have identified six memory traces that form in the olfactory nervous system after conditioning with odors. These traces occur in distinct nodes of the olfactory nervous system, form and disappear across different windows of time, and are detected in the imaged neurons as increased calcium influx or synaptic release in response to the conditioned odor. Three traces form at or near acquisition and coexist with short-term behavioral memory. One trace forms with a delay after learning and coexists with intermediate-term behavioral memory. Two traces form many hours after acquisition and coexist with long-term behavioral memory. The transient memory traces may support behavior across the time windows of their existence. The experimental approaches for dissecting memory formation in the fly, ranging from the molecular to the systems, make it an ideal system for elucidating the logic by which the nervous system organizes and stores different temporal forms of memory.     

Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output

Amnesiac mutant flies have an olfactory memory defect. The amn gene encodes a homolog of vertebrate pituitary adenylate cyclase-activating peptide (PACAP), and it is strongly expressed in dorsal paired medial (DPM) neurons. DPM neurons ramify throughout the mushroom bodies in the adult fly brain, and they are required for stable memory. Here, we show that DPM neuron output is only required during the consolidation phase for middle-term odor memory and is dispensable during acquisition and recall. However, we found that DPM neuron output is required during acquisition of a benzaldehyde odor memory. We show that flies sense benzaldehyde by the classical olfactory and a noncanonical route. These results suggest that DPM neurons are required to consolidate memory and are differently involved in memory of a volatile that requires multisensory integration.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

A pair of inhibitory neurons are required to sustain labile memory in the Drosophila mushroom body

Labile memory is thought to be held in the brain as persistent neural network activity. 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. DPM neurons innervate the entire MB lobe region and appear to be pre- and postsynaptic to the MB, consistent with a recurrent network model. Here we identify a role after training for synaptic output from the GABAergic anterior paired lateral (APL) neurons. 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 of 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.     

Parallel processing of appetitive short- and long-term memories in Drosophila

It is broadly accepted that long-term memory (LTM) is formed sequentially after learning and short-term memory (STM) formation, but the nature of the relationship between early and late memory traces remains heavily debated [1-5]. To shed light on this issue, we used an olfactory appetitive conditioning in Drosophila, wherein starved flies learned to associate an odor with the presence of sugar [6]. We took advantage of the fact that both STM and LTM are generated after a unique conditioning cycle [7, 8] to demonstrate that appetitive LTM is able to form independently of STM. More specifically, we show that (1) STM retrieval involves output from γ neurons of the mushroom body (MB), i.e., the olfactory memory center [9, 10], whereas LTM retrieval involves output from αβ MB neurons; (2) STM information is not transferred from γ neurons to αβ neurons for LTM formation; and (3) the adenylyl cyclase RUT, which is thought to operate as a coincidence detector between the olfactory stimulus and the sugar stimulus [11-14], is required independently in γ neurons to form appetitive STM and in αβ neurons to form LTM. Taken together, these results demonstrate that appetitive short- and long-term memories are formed and processed in parallel.     

Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation

Memories are formed, stabilized in a time-dependent manner, and stored in neural networks. In Drosophila, retrieval of punitive and rewarded odor memories depends on output from mushroom body (MB) neurons, consistent with the idea that both types of memory are represented there. Dorsal Paired Medial (DPM) neurons innervate the mushroom bodies, and DPM neuron output is required for the stability of punished odor memory. Here we show that stable reward-odor memory is also DPM neuron dependent. DPM neuron expression of amnesiac (amn) in amn mutant flies restores wild-type memory. In addition, disrupting DPM neurotransmission between training and testing abolishes reward-odor memory, just as it does with punished memory. We further examined DPM-MB connectivity by overexpressing a DScam variant that reduces DPM neuron projections to the MB alpha, beta, and gamma lobes. DPM neurons that primarily project to MB alpha' and beta' lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, our results suggest that the fly employs the local DPM-MB circuit to stabilize punitive- and reward-odor memories and that stable aspects of both forms of memory may reside in mushroom body alpha' and beta' lobe neurons.     

Dopamine is required for learning and forgetting in Drosophila

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose?a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.     

Parametric and genetic analysis of Drosophila appetitive long‐term memory and sugar motivation

Distinct forms of memory can be highlighted using different training protocols. In Drosophila olfactory aversive learning, one conditioning session triggers memory formation independently of protein synthesis, while five spaced conditioning sessions lead to the formation of long‐term memory (LTM), a long‐lasting memory dependent on de novo protein synthesis. In contrast, one session of odour–sugar association appeared sufficient for the fly to form LTM. We designed and tuned an apparatus that facilitates repeated discriminative conditioning by alternate presentations of two odours, one being associated with sugar, as well as a new paradigm to test sugar responsiveness (SR). Our results show that both SR and short‐term memory (STM) scores increase with starvation length before conditioning. The protein dependency of appetitive LTM is independent of the repetition and the spacing of training sessions, on the starvation duration and on the strength of the unconditioned stimulus. In contrast to a recent report, our test measures an abnormal SR of radish mutant flies, which might initiate their STM and LTM phenotypes. In addition, our work shows that crammer and tequila mutants, which are deficient for aversive LTM, present both an SR and an appetitive STM defect. Using the MB247‐P[switch] system, we further show that tequila is required in the adult mushroom bodies for normal sugar motivation.     

Insect olfactory memory in time and space

Recent studies using functional optical imaging have revealed that cellular memory traces form in different areas of the insect brain after olfactory classical conditioning. These traces are revealed as increased calcium signals or synaptic release from defined neurons, and include a short-lived trace that forms immediately after conditioning in antennal lobe projection neurons, an early trace in dopaminergic neurons, and a medium-term trace in dorsal paired medial neurons. New molecular genetic tools have revealed that for normal behavioral memory performance, synaptic transmission from the mushroom body neurons is required only during retrieval, whereas synaptic transmission from dopaminergic neurons is required at the time of acquisition and synaptic transmission from dorsal paired medial neurons is required during the consolidation period. Such experimental results are helping to identify the types of neurons that participate in olfactory learning and when their participation is required. Olfactory learning often occurs alongside crossmodal interactions of sensory information from other modalities. Recent studies have revealed complex interactions between the olfactory and the visual senses that can occur during olfactory learning, including the facilitation of learning about subthreshold olfactory stimuli due to training with concurrent visual stimuli.     

A persistent cellular change in a single modulatory neuron contributes to associative long-term memory

Most neuronal models of learning assume that changes in synaptic strength are the main mechanism underlying long-term memory (LTM) formation. However, we show here that a persistent depolarization of membrane potential, a type of cellular change that increases neuronal responsiveness, contributes significantly to a long-lasting associative memory trace. The use of a model invertebrate network with identified neurons and known synaptic connectivity had the advantage that the contribution of this cellular change to memory could be evaluated in a neuron with a known function in the learning circuit. Specifically, we used the well-understood motor circuit underlying molluscan feeding and showed that a key modulatory neuron involved in the initiation of feeding ingestive movements underwent a long-term depolarization following behavioral associative conditioning. This depolarization led to an enhanced single cell and network responsiveness to a previously neutral tactile conditioned stimulus, and the persistence of both matched the time course of behavioral associative memory. The change in the membrane potential of a key modulatory neuron is both sufficient and necessary to initiate a conditioned response in a reduced preparation and underscores its importance for associative LTM.     

Watching the fly brain learn

The peptidergic dorsal paired medial (DPM) neurons, which innervate the mushroom bodies in Drosophila, have been widely hypothesized to be part of the unconditioned stimulus (US) pathway of odor-shock classical conditioning. In the December 2 issue of Cell, Yu et al., using functional imaging techniques, report the surprising finding that DPMs contain odor-specific memory traces and send integrated information about the conditioned stimulus (CS) to the mushroom bodies. These findings provide important new insight into the circuitry of learning in Drosophila.     

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.     

桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

Contrasting Role of Octopamine in Appetitive and Aversive Learning in the Crab Chasmagnathus

Background

Biogenic amines are implicated in reinforcing associative learning. Octopamine (OA) is considered the invertebrate counterpart of noradrenaline and several studies in insects converge on the idea that OA mediates the reward in appetitive conditioning. However, it is possible to assume that OA could have a different role in an aversive conditioning.

Methodology/Principal Findings

Here we pharmacologically studied the participation of OA in two learning processes in the crab Chasmagnathus granulatus, one appetitive and one aversive. It is shown that the aversive memory is impaired by an OA injection applied immediately or 30 minutes after the last training trial. By contrast, the appetitive memory is blocked by OA antagonists epinastine and mianserine, but enhanced by OA when injected together with the supply of a minimum amount of reinforcement. Finally, double-learning experiments in which crabs are given the aversive and the appetitive learning either successively or simultaneously allow us to study the interaction between both types of learning and analyze the presumed action of OA. We found that the appetitive training offered immediately, but not one hour, after an aversive training has an amnesic effect on the aversive memory, mimicking the effect and the kinetic of an OA injection.

Conclusions/Significance

Our results demonstrate that the role of OA is divergent in two memory processes of opposite signs: on the one hand it would mediate the reinforcement in appetitive learning, and on the other hand it has a deleterious effect over aversive memory consolidation.     

Heterotypic gap junctions between two neurons in the drosophila brain are critical for memory

Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells. Although recent studies have linked gap junctions to memory formation, it remains unclear how they contribute to this process. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM.     

Molecular Signals into the Insular Cortex and Amygdala During Aversive Gustatory Memory Formation

In this paper, we will provide evidence of the putative molecular signals and biochemical events that mediate the formation of long-lasting gustatory memory trace. When an animal drinks a novel taste (the conditioned stimulus; CS) and it is later associated with malaise (unconditioned stimulus; US), the animal will reject it in the next presentation, developing a long-lasting taste aversion, i.e., the taste cue becomes an aversive signal, and this is referred to as conditioning taste aversion. Different evidence indicates that the novel stimulus (taste) induces a rapid and strong cortical acetylcholine activity that decreases when the stimulus becomes familiar after several presentations. Cholinergic activation via muscarinic receptors initiates a series of intracellular events leading to plastic changes that could be related to short- and/or long-term memory gustatory trace. Such plastic changes facilitate the incoming US signals carried out by, in part, the glutamate release induced by the US. Altogether, these events could produce the cellular changes related to the switch from safe to aversive taste memory trace. A proposed working model to explain the biochemical sequence of signals during taste memory formation will be discussed.     

Classical reward conditioning in Drosophila melanogaster

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.     

Nucleus accumbens neurons dynamically respond to appetitive and aversive associative learning

To survive, individuals must learn to associate cues in the environment with emotionally relevant outcomes. This association is partially mediated by the nucleus accumbens (NAc), a key brain region of the reward circuit that is mainly composed by GABAergic medium spiny neurons (MSNs), that express either dopamine receptor D1 or D2. Recent studies showed that both populations can drive reward and aversion, however, the activity of these neurons during appetitive and aversive Pavlovian conditioning remains to be determined. Here, we investigated the relevance of D1- and D2-neurons in associative learning, by measuring calcium transients with fiber photometry during appetitive and aversive Pavlovian tasks in mice. Sucrose was used as a positive valence unconditioned stimulus (US) and foot shock was used as a negative valence US. We show that during appetitive Pavlovian conditioning, D1- and D2-neurons exhibit a general increase in activity in response to the conditioned stimuli (CS). Interestingly, D1- and D2-neurons present distinct changes in activity after sucrose consumption that dynamically evolve throughout learning. During the aversive Pavlovian conditioning, D1- and D2-neurons present an increase in the activity in response to the CS and to the US (shock). Our data support a model in which D1- and D2-neurons are concurrently activated during appetitive and aversive conditioning.