Aversive Olfactory Learning Research Articles

Long-term memory requires sequential protein synthesis in three subsets of mushroom body output neurons in Drosophila

Creating long-term memory (LTM) requires new protein synthesis to stabilize learning-induced synaptic changes in the brain. In the fruit fly, Drosophila melanogaster, aversive olfactory learning forms several phases of labile memory to associate an odor with coincident punishment in the mushroom body (MB). It remains unclear how the brain consolidates early labile memory into LTM. Here, we survey 183 Gal4 lines containing almost all 21 distinct types of MB output neurons (MBONs) and show that sequential synthesis of learning-induced proteins occurs at three types of MBONs. Downregulation of oo18 RNA-binding proteins (ORBs) in any of these MBONs impaired LTM. And, neurotransmission outputs from these MBONs are all required during LTM retrieval. Together, these results suggest an LTM consolidation model in which transient neural activities of early labile memory in the MB are consolidated into stable LTM at a few postsynaptic MBONs through sequential ORB-regulated local protein synthesis.

Tunicamycin impairs olfactory learning and synaptic plasticity in the olfactory bulb

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1μM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

Developmentally defined forebrain circuits regulate appetitive and aversive olfactory learning.

Postnatal and adult neurogenesis are region- and modality-specific, but the significance of developmentally distinct neuronal populations remains unclear. We demonstrate that chemogenetic inactivation of a subset of forebrain and olfactory neurons generated at birth disrupts responses to an aversive odor. In contrast, novel appetitive odor learning is sensitive to inactivation of adult born neurons, unveiling that developmentally defined sets of neurons may differentially participate in hedonic aspects of sensory learning.

Caenorhabditis elegans employs innate and learned aversion in response to bacterial toxic metabolites tambjamine and violacein.

Bacteriovorus eukaryotes such as nematodes are one of the major natural predators of bacteria. In their defense bacteria have evolved a number of strategies to avoid predation, including the production of deterrent or toxic metabolites, however little is known regarding the response of predators towards such bacterial defenses. Here we use the nematode C. elegans as a model to study a predators’ behavioral response towards two toxic bacterial metabolites, tambjamine YP1 and violacein. We found that C. elegans displays an innate avoidance behavior towards tambjamine YP1, however requires previous exposure to violacein before learning to avoid this metabolite. The learned avoidance of violacein is specific, reversible, is mediated via the nematode olfactory apparatus (aversive olfactory learning) and is reduced in the absence of the neurotransmitter serotonin. These multiple strategies to evade bacterial toxic metabolites represent a valuable behavioral adaptation allowing bacteriovorus predators to distinguish between good and bad food sources, thus contributing to the understanding of microbial predator-prey interactions.

Parallel encoding of sensory history and behavioral preference during Caenorhabditis elegans olfactory learning.

Sensory experience modifies behavior through both associative and non-associative learning. In Caenorhabditis elegans, pairing odor with food deprivation results in aversive olfactory learning, and pairing odor with food results in appetitive learning. Aversive learning requires nuclear translocation of the cGMP-dependent protein kinase EGL-4 in AWC olfactory neurons and an insulin signal from AIA interneurons. Here we show that the activity of neurons including AIA is acutely required during aversive, but not appetitive, learning. The AIA circuit and AGE-1, an insulin-regulated PI3 kinase, signal to AWC to drive nuclear enrichment of EGL-4 during conditioning. Odor exposure shifts the AWC dynamic range to higher odor concentrations regardless of food pairing or the AIA circuit, whereas AWC coupling to motor circuits is oppositely regulated by aversive and appetitive learning. These results suggest that non-associative sensory adaptation in AWC encodes odor history, while associative behavioral preference is encoded by altered AWC synaptic activity.

Optogenetics in Drosophila Neuroscience.

Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory.

The NMDA Receptor Promotes Sleep in the Fruit Fly, Drosophila melanogaster.

Considerable evidence indicates that sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. We previously found a short sleeper mutant, fumin (fmn), and identified its mutation in the dopamine transporter gene. We reported similarities in the molecular basis of sleep and arousal regulation between mammals and Drosophila. In aversive olfactory learning tasks, fmn mutants demonstrate defective memory retention, which suggests an association between sleep and memory. In an attempt to discover additional sleep related genes in Drosophila, we carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes are differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, we performed a functional screen. We found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. The NMDA receptor (NMDAR) plays an important role in learning and memory both in Drosophila and mammals. The application of the NMDAR antagonist, MK-801, reduced sleep in control flies, but not in fmn. These results suggest that NMDAR promotes sleep regulation in Drosophila.

Commentary: Olfactory aversive conditioning during sleep reduces cigarette-smoking behavior.

Commentary: Olfactory aversive conditioning during sleep reduces cigarette-smoking behavior.

The neonicotinoid imidacloprid impairs honey bee aversive learning of simulated predation.

Neonicotinoid insecticides can impair bee learning and memory--cognitive features that play a key role in colony fitness because they facilitate foraging. For example, the commonly used neonicotinoid imidacloprid reduces honey bee olfactory learning. However, no studies have previously determined whether imidacloprid can impair aversive associative learning, although such learning should enhance bee survival by allowing bees to avoid dangerous foraging sites. To mimic attempted predation of foragers, we developed an electro-mechanical predator that consistently attacked foragers with a pinching bite at a fixed force and elicited aversive olfactory learning in a sting extension response (SER) assay. We show that chronic exposure to a sublethal concentration of imidacloprid (25.6 µg l(-1)=20.8 ppb) over 4 days (mean of 1.5 ng per bee day(-1)), significantly impaired aversive short-term learning and memory retention. Imidacloprid treatment reduced short-term learning by 87% and memory retention by 85% in comparison with control bees. Imidacloprid therefore impairs the ability of honey bees to associate a naturalistic predation stimulus--biting--with floral odor compounds. Such learning should enhance bee survival, suggesting that xenobiotics could alter more complex ecological interactions such as predator-prey relationships.

Olfactory preference conditioning changes the reward value of reinforced and non-reinforced odors.

Olfaction is determinant for the organization of rodent behavior. In a feeding context, rodents must quickly discriminate whether a nutrient can be ingested or whether it represents a potential danger to them. To understand the learning processes that support food choice, aversive olfactory learning and flavor appetitive learning have been extensively studied. In contrast, little is currently known about olfactory appetitive learning and its mechanisms. We designed a new paradigm to study conditioned olfactory preference in rats. After 8 days of exposure to a pair of odors (one paired with sucrose and the other with water), rats developed a strong and stable preference for the odor associated with the sucrose solution. A series of experiments were conducted to further analyze changes in reward value induced by this paradigm for both stimuli. As expected, the reward value of the reinforced odor changed positively. Interestingly, the reward value of the alternative odor decreased. This devaluation had an impact on further odor comparisons that the animal had to make. This result suggests that appetitive conditioning involving a comparison between two odors not only leads to a change in the reward value of the reinforced odor, but also induces a stable devaluation of the non-reinforced stimulus.

Male cognitive performance declines in the absence of sexual selection

Sexual selection is responsible for the evolution of male ornaments and armaments, but its role in the evolution of cognition--the ability to process, retain and use information--is largely unexplored. Because successful courtship is likely to involve processing information in complex, competitive sexual environments, we hypothesized that sexual selection contributes to the evolution and maintenance of cognitive abilities in males. To test this, we removed mate choice and mate competition from experimental populations of Drosophila melanogaster by enforcing monogamy for over 100 generations. Males evolved under monogamy became less proficient than polygamous control males at relatively complex cognitive tasks. When faced with one receptive and several unreceptive females, polygamous males quickly focused on receptive females, whereas monogamous males continued to direct substantial courtship effort towards unreceptive females. As a result, monogamous males were less successful in this complex setting, despite being as quick to mate as their polygamous counterparts with only one receptive female. This diminished ability to use past information was not limited to the courtship context: monogamous males (but not females) also showed reduced aversive olfactory learning ability. Our results provide direct experimental evidence that the intensity of sexual selection is an important factor in the evolution of male cognitive ability.

The olfactory neuron AWC promotes avoidance to normally palatable food following chronic dietary restriction

Changes in metabolic state alter foraging behavior and food preference in animals. Here, I show that normally attractive food becomes repulsive to Caenorhabditis elegans if animals are chronically undernourished as a result of alimentary tract defects. This behavioral plasticity is achieved in two ways: increased food leaving and induction of aversive behavior towards food. A particularly strong food avoider is defective in the chitin synthase that makes the pharyngeal lining. Food avoidance induced by underfeeding is mediated by cGMP signaling in the olfactory neurons AWC and AWB, and the gustatory neurons ASJ and ASK. Food avoidance is enhanced by increased population density and is reduced if the animals are unable to correctly interpret their nutritional state as a result of defects in the AMP kinase or TOR/S6kinase pathways. The TGF-β/DBL-1 pathway suppresses food avoidance and the cellular basis for this is distinct from its role in aversive olfactory learning of harmful food. This study suggests that nutritional state feedback via nutrient sensors, population size and olfactory neurons guides food preference in C. elegans.

Appetitive and aversive olfactory learning induce similar generalization rates in the honey bee

Appetitive and aversive learning drive an animal toward or away from stimuli predicting reinforcement, respectively. The specificity of these memories may vary due to differences in cost–benefit relationships associated with appetitive and aversive contexts. As a consequence, generalization performances may differ after appetitive and aversive training. Here, we determined whether honey bees show different rates of olfactory generalization following appetitive olfactory conditioning of the proboscis extension response, or aversive olfactory conditioning of the sting extension response. In both cases, we performed differential conditioning, which improves discrimination learning between a reinforced odor (CS?) and a non-reinforced odor (CS-) and evaluated generalization to two novel odors whose similarity to the CS? and the CS- was different. We show, given the same level of discriminatory performance, that rates of generalization are similar between the two conditioning protocols and discuss the possible causes for this phenomenon.

Two Insulin-like Peptides Antagonistically Regulate Aversive Olfactory Learning in C. elegans

The insulin/insulin-like peptides (ILPs) regulate key events in physiology, including neural plasticity. However, the cellular and circuit mechanisms whereby ILPs regulate learning remain largely unknown. Here, we characterize two ILPs that play antagonistic roles in aversive olfactory learning of C. elegans. We show that the ILP ins-6 acts from ASI sensory neurons to enable learning by repressing the transcription of another ILP, ins-7, specifically in URX neurons. A high level of INS-7 from URX disrupts learning by antagonizing the insulin receptor-like homolog DAF-2 in the postsynaptic neurons RIA, which play an essential role in the neural circuit underlying olfactory learning. We also show that increasing URX-generated INS-7 and loss of INS-6, both of which abolish learning, alter RIA neuronal property. Together, our results reveal an "ILP-to-ILP" pathway that links environment-sensing neurons, ASI and URX, to the key neuron, RIA, of a network that underlies olfactory plasticity and modulates its activity.

Histone acetylation in the olfactory bulb of young rats facilitates aversive olfactory learning and synaptic plasticity

Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

High-Resolution Linkage Analyses to Identify Genes That Influence Varroa Sensitive Hygiene Behavior in Honey Bees

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene ‘no receptor potential A’ and a dopamine receptor. ‘No receptor potential A’ is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

DBL-1, a TGF-β, is essential for Caenorhabditis elegans aversive olfactory learning

The TGF-β superfamily is conserved throughout metazoan, and its members play essential roles in development and disease. TGF-β has also been implicated in adult neural plasticity. However, the underlying mechanisms are not well understood. Here we report that DBL-1, a Caenorhabditis elegans TGF-β homolog known to control body morphology and immunity, is essential for aversive olfactory learning of potentially harmful bacteria food. We show that DBL-1 generated by the AVA command interneurons, which are critical for sensorimotor responses, regulates aversive olfactory learning, and that the activity of the type I TGF-β receptor SMA-6 in the hypodermis is needed during adulthood to generate olfactory plasticity. These spatial and temporal mechanisms are critical for the DBL-1 signaling to achieve its diverse functions in development and adult neural plasticity. Interestingly, aversive training decreases AVA calcium response, leading to an increase in the DBL-1 signal secreted from AVA, revealing an experience-dependent change that can underlie the role of TGF-β signaling in mediating plasticity.

A role for α-adducin (ADD-1) in nematode and human memory

Identifying molecular mechanisms that underlie learning and memory is one of the major challenges in neuroscience. Taken the advantages of the nematode Caenorhabditis elegans, we investigated α-adducin (add-1) in aversive olfactory associative learning and memory. Loss of add-1 function selectively impaired short- and long-term memory without causing acquisition, sensory, or motor deficits. We showed that α-adducin is required for consolidation of synaptic plasticity, for sustained synaptic increase of AMPA-type glutamate receptor (GLR-1) content and altered GLR-1 turnover dynamics. ADD-1, in a splice-form- and tissue-specific manner, controlled the storage of memories presumably through actin-capping activity. In support of the C. elegans results, genetic variability of the human ADD1 gene was significantly associated with episodic memory performance in healthy young subjects. Finally, human ADD1 expression in nematodes restored loss of C. elegans add-1 gene function. Taken together, our findings support a role for α-adducin in memory from nematodes to humans. Studying the molecular and genetic underpinnings of memory across distinct species may be helpful in the development of novel strategies to treat memory-related diseases.

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.

Aversive olfactory learning and associative long-term memory in Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) adult hermaphrodite has 302 invariant neurons and is suited for cellular and molecular studies on complex behaviors including learning and memory. Here, we have developed protocols for classical conditioning of worms with 1-propanol, as a conditioned stimulus (CS), and hydrochloride (HCl) (pH 4.0), as an unconditioned stimulus (US). Before the conditioning, worms were attracted to 1-propanol and avoided HCl in chemotaxis assay. In contrast, after massed or spaced training, worms were either not attracted at all to or repelled from 1-propanol on the assay plate. The memory after the spaced training was retained for 24 h, while the memory after the massed training was no longer observable within 3 h. Worms pretreated with transcription and translation inhibitors failed to form the memory by the spaced training, whereas the memory after the massed training was not significantly affected by the inhibitors and was sensitive to cold-shock anesthesia. Therefore, the memories after the spaced and massed trainings can be classified as long-term memory (LTM) and short-term/middle-term memory (STM/MTM), respectively. Consistently, like other organisms including Aplysia, Drosophila, and mice, C. elegans mutants defective in nmr-1 encoding an NMDA receptor subunit failed to form both LTM and STM/MTM, while mutations in crh-1 encoding the CREB transcription factor affected only the LTM.