Single Conditioning Trial Research Articles

Yellowtail damselfish Chrysiptera parasema can associate predation risk with the acoustic call of a heterospecific damselfish following pairing with conspecific alarm cues.

The ability to detect and respond to the presence of predation risk is under intense selection, especially for small-bodied fishes. Damselfishes (Pomacentridae) use auditory vocalizations during inter- and intrasexual interactions, but it is not known if they can use vocalizations in the context of predator-prey interactions. Here, we test if yellowtail damselfish, Chrysiptera parasema, can learn to associate the territorial vocalization of heterospecific humbug damselfish Dascyllus aruanus with predation risk. In conditioning trials yellowtail damselfish were presented with the territorial call of humbug damselfish while either blank water (control treatment) or chemical alarm cue derived from damaged skin of conspecific yellowtail damselfish was introduced. In conditioning trials, fish exposed to alarm cue exhibited increased activity and spent more time in the water column relative to fish that received the control treatment. After a single conditioning trial, conditioned fish were exposed again to the territorial call of humbug damselfish. Fish conditioned with the call + alarm cue showed increased activity and spent more time in the water column relative to fish that had been conditioned with the control treatment. These data indicate associative learning of an auditory stimulus with predation risk in a species that regularly uses auditory signalling in other contexts. Recordings of conditioning and test trials failed to detect any acoustic calls produced by test fish in response to the perception of predation risk. Thus, although yellowtail damselfish can associate risk with auditory stimuli, we found no evidence that they produce an alarm call.

The role of cAMP-dependent protein kinase A in the formation of long-term memory in Bactrocera dorsalis

The cAMP-dependent protein kinase A (PKA) signaling pathway has long been considered critical for long-term memory (LTM) formation. Previous studies have mostly focused on the role of PKA signaling in LTM induction by multiple spaced conditioning with less attention to LTM induction by a single conditioning. Here, we conducted behavioral-pharmacology, enzyme immunoassay and RNA interference experiments to study the role of the PKA signaling pathway in LTM formation in the agricultural pest Bactrocera dorsalis, which has a strong memory capacity allowing it to form a two-day memory even from a single conditioning trial. We found that either blocking or activating PKA prior to conditioning pretreatment affected multiple spaced LTM, and conversely, they did not affect LTM formed by single conditioning. This was further confirmed by enzyme-linked immunosorbent assay (ELISA) and silencing of the protein kinase regulatory subunit 2 and catalytic subunit 1. Taken together, these results suggest that activating PKA during memory acquisition helps to induce the LTM formed by multiple spaced conditioning but not by a single conditioning. Our findings challenge the conserved role of PKA signaling in LTM, which provides a basis for the greater diversity of molecular mechanisms underlying LTM formation across species, as well as possible functional and evolutionary implications.

Genetic and environmental influences on one-trial conditioned context aversion in mice.

Anticipatory nausea (AN) is caused by an association between contextual cues and the experience of nausea (the side effects of chemotherapy or radiation treatment) and it develops predominantly in female patients undergoing chemotherapy. Preclinical studies in rodents show that the administration of an illness-inducing agent in the presence of novel contextual cues can cause conditioned context aversion (CCA) and this has been proposed to model AN. The literature also suggests that brief pre-exposure to a novel context prior to shock delivery is critical in the development of contextual fear conditioning in rodents (a phenomenon known as Immediate Shock Deficit), but this has not been assessed in CCA. The aim of present study was to develop a CCA paradigm to assess this in outbred (CD1) and inbred (C57BL/6J) mice and evaluate potential sex differences. The results revealed that a single conditioning trial in which a distinctive context was paired with LiCl-induced illness was sufficient to elicit a conditioned response in both female and male CD1 outbred mice, but not in C57BL/6J inbred mice. In addition, CCA was facilitated when animals had prior experience with the context. Finally, outbred female mice showed longer and more robust retention of CCA than male mice, which parallels clinical findings. The results indicate the importance of using CD1 outbred mice as an animal model of AN as well as examining sex differences in the CCA paradigm. Similar findings in humans encourage the future use of this novel CCA preclinical mouse model.

Formation characteristics of long-term memory in Bactrocera dorsalis.

Studies on insects have contributed significantly to a better understanding of learning and memory, which is a necessary cognitive capability for all animals. Although the formation of memory has been studied in some model insects, more evidence is required to clarify the characteristics of memory formation, especially long-term memory (LTM), which is important for reliably storing information. Here, we explored this question by examining Bactrocera dorsalis, an agricultural pest with excellent learning abilities. Using the classical conditioning paradigm of the olfactory proboscis extension reflex (PER), we found that paired conditioning with multiple trials (>3) spaced with an intertrial interval (≥10min) resulted in stable memory that lasted for at least 3d. Furthermore, even a single conditioning trial was sufficient for the formation of a 2-d memory. With the injection of protein inhibitors, protein-synthesis-dependent memory was confirmed to start 4h after training, and its dependence on translation and transcription differed. Moreover, the results revealed that the dependence of memory on protein translation exhibited a time-window effect (4-6h). Our findings provide an integrated view of LTM in insects, suggesting common mechanisms in LTM formation that play a key role in the biological basis of memory.

Sex Differences in Cue Competition Effects With a Conditioned Taste Aversion Preparation.

This study aimed to test whether male and female rats might show differences in cue competition effects in a conditioned taste aversion (CTA) model. Experiment 1 tested for sex differences in overshadowing. After conditioning of a flavored compound AB or only one simple flavor A (being A and B a solution of sugar 10% and salt 1%, counterbalanced), consumption of the A solution at test was larger in the former than in the latter case only in males. Thus, the usual effect of overshadowing was observed in males but not in females. Experiment 2 examined sex differences in blocking with the same stimuli used in Experiment 1. After conditioning of AB, the consumption of B was larger for the animals that previously received a single conditioning trial with A than for those that received unpaired presentations of A and the illness. As observed in Experiment 1, the typical blocking effect appeared only in males but not in females. The present findings thus support the hypothesis that sex dimorphism might be expressed in classical conditioning, or at least, in cue competition effects such as overshadowing and blocking with a taste aversion model.

Redefining Single-Trial Memories in the Honeybee

Research on honeybee memory has led to a widely accepted model in which a single pairing of an odor stimulus with sucrose induces memories that are independent of protein synthesis but is unable to form protein-synthesis-dependent long-term memory (LTM). The latter is said to arise only after three or more pairings of odor and sucrose. Here, we show that this model underestimates the capacity of the bee brain to form LTMs after a unique appetitive experience. Using state-of-the art conditioning setups and individual-based analyses of conditioned responses, we found that protein-synthesis-dependent memories are formed already 4h after the single conditioning trial and persist even 3days later. These memories (4 h, 24 h, and 72 h) exhibit different dependencies on transcription and translation processes. Our results thus modify the traditional view of one-trial memories in an insect with a model status for memory research.

Ants learn fast and do not forget: associative olfactory learning, memory and extinction in Formica fusca.

Learning is a widespread phenomenon that allows behavioural flexibility when individuals face new situations. However, learned information may lose its value over time. If such a memory endures, it can be deleterious to individuals. The process of extinction allows memory updating when the initial information is not relevant anymore. Extinction is widespread among animals, including humans. We investigated associative appetitive learning in an ant species that is widely distributed in the Northern Hemisphere, Formica fusca. We studied acquisition and memory between 1 h and one week after conditioning, as well as the extinction process. Ants learn very rapidly, their memory lasts up to 3 days, decreases slowly over time and is highly resistant to extinction, even after a single conditioning trial. Using a pharmacological approach, we show that this single-trial memory critically depends on protein synthesis (long-term memory). These results indicate that individual ant workers of F. fusca show remarkable learning and memory performances. Intriguingly, they also show a strong resistance to updating learned associations. Resistance to extinction may be advantageous when the environment is stochastic and individuals need to switch often from one learned task to another.

Subthreshold Fear Conditioning Produces a Rapidly Developing Neural Mechanism that Primes Subsequent Learning.

Learning results in various forms of neuronal plasticity that provide a lasting representation of past events, and understanding the mechanisms supporting lasting memories has been a primary pursuit of the neurobiological study of memory. However, learning also alters the capacity for future learning, an observation that likely reflects its adaptive significance. In the laboratory, we can study this essential property of memory by assessing how prior experience alters the capacity for subsequent learning. Previous studies have indicated that while a single weak fear conditioning trial is insufficient to support long-term memory (LTM), it can facilitate future learning such that another trial delivered within a protracted time window results in a robust memory. Here, we sought to determine whether or not manipulating neural activity in the basolateral amygdala (BLA) using designer receptors exclusively activated by designer drugs (DREADDs) during or after the initial learning trial would affect the ability of the initial trial to facilitate subsequent learning. Our results show that inhibiting the BLA in rats prior to the first trial prevented the ability of that trial to facilitate learning when a second trial was presented the next day. Inhibition of the BLA immediately after the first trial using DREADDs was not effective, nor was pharmacological inhibition of protein kinase A (PKA) or the mitogen-activated protein kinase (MAPK). These findings indicate that the neural mechanisms that permit an initial subthreshold fear conditioning trial to alter later learning develop rapidly and do not appear to require a typical post-learning consolidation period.

Effect of Chronic Inflammatory Pain on Rewarding Effects of Morphine in Male versus Female Rats

Morphine and other opioid analgesics are widely used to treat both acute and chronic pain conditions. While they are effective pain‐relievers, their action at the μ‐opioid receptor means that they are also rewarding, and their use can lead to addiction. Previous research indicates that pain may reduce the rewarding effects of morphine. In this study, we sought to compare morphine potency and efficacy to produce a conditioned place preference (CPP, a measure of reward) between rats that were in chronic pain and rats that were not. Because sex differences in morphine's rewarding and antinociceptive effects have been demonstrated, both male and female rats were conditioned using a three‐day conditioning paradigm. On day one, rats could freely explore both sides of the two‐sided conditioning apparatus. The sides differed in color (black versus white walls) and floor type (bars versus wire grid); the sides were divided by a small section of smooth metal on which the animal was initially placed. Time spent on each side of the apparatus and number of crosses between sides in 15 min were recorded. Then mineral oil vehicle or Complete Freund's Adjuvant (CFA) was injected into one hind paw. On day two, rats underwent two conditioning trials 6 h apart wherein morphine treatment was paired with one side of the apparatus and saline was paired with the other. Morphine was administered s.c. at doses of 0.0, 0.1, 0.32, 1.0, 3.2, 5.6 or 10.0 mg/kg to male and female rats (and 18.0 mg/kg was tested in females only), and then rats were immediately placed into one side of the apparatus for 30 min. Saline was administered before the second conditioning trial, after which rats were restricted to the opposite side of the apparatus for 30 min. On the third day, rats could explore the entire apparatus for 15 min, and time spent on each side, midline crosses, and paw thickness were recorded. Preliminary data show that, consistent with previous research, morphine treatment does not affect CFA‐induced inflammation. However, in both male and female rats, a single conditioning trial with morphine dose‐dependently induced place preference. Compared to control rats treated with intraplantar mineral oil, the peak place preference in CFA‐treated rats was shifted toward lower doses of morphine – however, morphine CPP varied significantly by the side of the apparatus that was paired with morphine, particularly in CFA‐treated rats. Specifically, greater place preference was observed when morphine was paired with the black wall/grid floor side than with the white wall/bar floor side. With approximately 75% of rats tested, no sex differences are apparent. These results suggest that rats with inflammatory pain experience rewarding effects from morphine differently than do healthy animals. Clinically, these results are of interest because they suggest that morphine's rewarding effects are not only affected by pain, but also by environment/context, particularly when subjects are in chronic pain.Support or Funding InformationSupported by The Psychopharmacology FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sexually experienced, but not naïve, female rats show a conditioned object preference (COP) for mating after a single training trial

Female rats with mating experience spend more time with the male rat, exhibit shorter contact-return latency to intromission, and display more proceptive behaviors in the male rat's compartment than during the first mating experience. The present study tested 1) whether mating induced conditioned object preference (COP) is possible with a single conditioning trial and 2) whether a preference is induced for an object associated with the first mating encounter or the fifth mating encounter in female rats. Ovariectomized, Long-Evans female rats were primed with estradiol benzoate + progesterone and either exposed to an empty paced mating chamber for 15 min (Naïve) or received a 15 intromission test of paced mating behavior (Experienced) on four separate occasions before undergoing the COP procedure. Experienced, but not Naïve, female rats developed a COP for a single mating bout, indicating that mating is highly rewarding for sexually experienced female rats. The findings raise questions about the effect of sexual experience on reward regions in the brain, the responsiveness of genital tissue, and learning mechanisms.

A CREB2-targeting microRNA is required for long-term memory after single-trial learning

Although single-trial induced long-term memories (LTM) have been of major interest in neuroscience, how LTM can form after a single episode of learning remains largely unknown. We hypothesized that the removal of molecular inhibitory constraints by microRNAs (miRNAs) plays an important role in this process. To test this hypothesis, first we constructed small non-coding RNA (sncRNA) cDNA libraries from the CNS of Lymnaea stagnalis subjected to a single conditioning trial. Then, by next generation sequencing of these libraries, we identified a specific pool of miRNAs regulated by training. Of these miRNAs, we focussed on Lym-miR-137 whose seed region shows perfect complementarity to a target sequence in the 3’ UTR of the mRNA for CREB2, a well-known memory repressor. We found that Lym-miR-137 was transiently up-regulated 1 h after single-trial conditioning, preceding a down-regulation of Lym-CREB2 mRNA. Furthermore, we discovered that Lym-miR-137 is co-expressed with Lym-CREB2 mRNA in an identified neuron with an established role in LTM. Finally, using an in vivo loss-of-function approach we demonstrated that Lym-miR-137 is required for single-trial induced LTM.

Acquisition and retention of conditioned aversions to context and taste in laboratory mice.

We compared the rate of acquisition and strength of retention of conditioned context aversion (CCA) with conditioned taste aversion (CTA) using pigmented, genetically heterogeneous mice (derived from Large and Small strains). Extending previous findings, in Experiment 1, mice accustomed to drinking from large glass bottles in the colony room learned to avoid graduated tubes after a single conditioning trial when drinking from these novel tubes was paired with injections of LiCl. The results also showed that CCA could be developed even when there was a 30-minute delay between conditioned stimulus and unconditioned stimulus. Retention of the aversion lasted for 4 weeks in both Immediate and Delay groups. Studies of conditioned saccharin aversion were conducted in Experiment 2. CTA acquisition was very similar to that observed in CCA and duration of aversion retention was similar in the CCA and CTA Delay groups, although at least 2 weeks longer in the Immediate group. Thus, CCA acquisition and retention characteristics are closer to those seen for CTA than has previously been reported. In Experiment 3, we examined whether albino mice (which are known to have weaker visual abilities compared to pigmented mice) would develop CCA comparable to those of pigmented mice. The development of conditioned aversion and its duration of retention was similar in albinos and pigmented mice. Nonspecific aversion emerged as an important contributor to strength of aversion during retention trials in both CCA and CTA paradigms with pigmented (but not albino) mice and deserves additional scrutiny in this field of inquiry.

One-trial overshadowing: Evidence for fast specific fear learning in humans

Adaptive defensive actions necessitate a fear learning system that is both fast and specific. Fast learning serves to minimize the number of threat confrontations, while specific learning ensures that the acquired fears are tied to threat-relevant cues only. In Pavlovian fear conditioning, fear acquisition is typically studied via repetitive pairings of a single cue with an aversive experience, which is not optimal for the examination of fast specific fear learning. In this study, we adopted the one-trial overshadowing procedure from basic learning research, in which a combination of two visual cues is presented once and paired with an aversive electrical stimulation. Using on-line shock expectancy ratings, skin conductance reactivity and startle reflex modulation as indices of fear learning, we found evidence of strong fear after a single conditioning trial (fast learning) as well as attenuated fear responding when only half of the trained stimulus combination was presented (specific learning). Moreover, specificity of fear responding tended to correlate with levels of state and trait anxiety. These results suggest that one-trial overshadowing can be used as a model to study fast specific fear learning in humans and individual differences therein.

Mechanisms underlying long-term fear memory formation from a metaplastic neuronal state

We previously showed that a single weak fear conditioning trial, that does not produce a long-term fear memory (LTM), appeared to prime memory formation such that when a second trial followed within a circumscribed time window a robust and long-lasting fear memory was formed. We also showed that this priming effect could be blocked if we interfered with protein kinase A (PKA) signaling in the amygdala during the first conditioning trial. The goals of the current study were to determine if LTM formation after the second trial depends on PKA signaling in the amygdala and to characterize the underlying memory processes engaged during the second trial that allows for LTM formation. Our interpretation of the original findings is that the second conditioning trial triggers LTM from a metaplastic state that is engaged by the first conditioning trial. However, it is also possible that the second conditioning trial acts as a reminder of the first and engages a reconsolidation-like process. Several experiments were conducted to distinguish between these two possibilities. We show that interfering with PKA signaling during the second conditioning trial disrupts memory formation. However, if a third trial follows the second or if the second trial was presented without shock, the PKA inhibitor was no longer effective. Our findings demonstrate that the induction of fear memory from a metaplastic state involves new learning that is distinct from retrieval-dependent updating of memories.

Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats.

The number and subtype composition of N-methyl-d-aspartate receptor (NMDAR) at synapses determines their functional properties and role in learning and memory. Genetically increased or decreased amount of GluN2B affects hippocampus-dependent memory in the adult brain. But in some experimental conditions (e.g., memory elicited by a single conditioning trial (1 CS-US)), GluN2B is not a necessary factor, which indicates that the precise role of GluN2B in memory formation requires further exploration. Here, we examined the role of GluN2B in the consolidation of fear memory using two training paradigms. We found that GluN2B was only required for the consolidation of memory elicited by five conditioning trials (5 CS-US), not by 1 CS-US. Strikingly, the expression of membrane GluN2B in CA1was training-strength-dependently increased after conditioning, and that the amount of membrane GluN2B determined its involvement in memory consolidation. Additionally, we demonstrated the increases in the activities of cAMP, ERK, and CREB in the CA1 after conditioning, as well as the enhanced intrinsic excitability and synaptic efficacy in CA1 neurons. Up-regulation of membrane GluN2B contributed to these enhancements. These studies uncover a novel mechanism for the involvement of GluN2B in memory consolidation by its accumulation at the cell surface in response to behavioral training.

Conditioned context aversion learning in the laboratory mouse.

It is well known that pairing of large contextual changes with illness can cause conditioned context aversion in laboratory rats. The aim of present study was to develop a paradigm to study this phenomenon in laboratory mice, a species widely employed in neurobehavioral studies. Genetically heterogeneous mice, drinking from plastic bottles in the colony room, learned to avoid glass bottles after a single conditioning trial when drinking from these was paired with injections of lithium chloride. The aversion was independent of any difference in the taste of water in plastic vs. glass bottles. When the variation in the visual stimulus was less distinct, development of a strong aversion required two conditioning trials and was not retained as well. The results also showed that conditioned context aversion, just like conditioned taste aversion, could also be developed across a 30-minute CS-UCS delay. The fact that taste was not a factor in distinguishing drinking from glass and plastic water bottles raises the possibility that, contextual stimuli, not taste, may have been the CS when rats (in Garcia's original experiments) avoided drinking from plastic bottles that had been paired with radiation. The development of contextual aversion conditioning protocols for mice will enable the molecular resources available for this species to be exploited. Furthermore, representation of the CS by discrete rather than the multimodal CSs typically used in most studies on contextual conditioning offers more focus when considering its neuroanatomical basis.

Proximal, but not distal, pre-exposure reduces serial overshadowing in one-trial taste aversion learning

This experiment tested whether pre-exposing a taste would reduce its ability to overshadow conditioning to a target taste and whether this effect would depend on the delay between pre-exposure and conditioning. Two groups of rats were pre-exposed to an interfering taste (HCl) either a week before conditioning (Group Distal) or the day preceding conditioning (Group Proximal). In the single conditioning trial, rats were given the target taste (sucrose) and 65min later were injected with lithium. The groups differed as to what they were given to drink 50min after sucrose: The Distal, Proximal and Novel groups were given HCl, while the Control group was given water. Pre-exposure to HCl reduced overshadowing of the sucrose aversion by HCl in Group Proximal but not in Group Distal. Possible explanations for the latter result include extinction of the context-HCl association and loss of context control over an HCl-no outcome association.

Learning-induced gene expression in the heads of two Nasonia species that differ in long-term memory formation.

BackgroundCellular processes underlying memory formation are evolutionary conserved, but natural variation in memory dynamics between animal species or populations is common. The genetic basis of this fascinating phenomenon is poorly understood. Closely related species of Nasonia parasitic wasps differ in long-term memory (LTM) formation: N. vitripennis will form transcription-dependent LTM after a single conditioning trial, whereas the closely-related species N. giraulti will not. Genes that were differentially expressed (DE) after conditioning in N. vitripennis, but not in N. giraulti, were identified as candidate genes that may regulate LTM formation.ResultsRNA was collected from heads of both species before and immediately, 4 or 24 hours after conditioning, with 3 replicates per time point. It was sequenced strand-specifically, which allows distinguishing sense from antisense transcripts and improves the quality of expression analyses. We determined conditioning-induced DE compared to naïve controls for both species. These expression patterns were then analysed with GO enrichment analyses for each species and time point, which demonstrated an enrichment of signalling-related genes immediately after conditioning in N. vitripennis only. Analyses of known LTM genes and genes with an opposing expression pattern between the two species revealed additional candidate genes for the difference in LTM formation. These include genes from various signalling cascades, including several members of the Ras and PI3 kinase signalling pathways, and glutamate receptors. Interestingly, several other known LTM genes were exclusively differentially expressed in N. giraulti, which may indicate an LTM-inhibitory mechanism. Among the DE transcripts were also antisense transcripts. Furthermore, antisense transcripts aligning to a number of known memory genes were detected, which may have a role in regulating these genes.ConclusionThis study is the first to describe and compare expression patterns of both protein-coding and antisense transcripts, at different time points after conditioning, of two closely related animal species that differ in LTM formation. Several candidate genes that may regulate differences in LTM have been identified. This transcriptome analysis is a valuable resource for future in-depth studies to elucidate the role of candidate genes and antisense transcription in natural variation in LTM formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1355-1) contains supplementary material, which is available to authorized users.

Introgression study reveals two quantitative trait loci involved in interspecific variation in memory retention among Nasonia wasp species.

Genes involved in the process of memory formation have been studied intensively in model organisms; however, little is known about the mechanisms that are responsible for natural variation in memory dynamics. There is substantial variation in memory retention among closely related species in the parasitic wasp genus Nasonia. After a single olfactory conditioning trial, N. vitripennis consolidates long-term memory that lasts at least 6 days. Memory of the closely related species N. giraulti is present at 24 h but is lost within 2 days after a single trial. The genetic basis of this interspecific difference in memory retention was studied in a backcrossing experiment in which the phenotype of N. giraulti was selected for in the background of N. vitripennis for up to five generations. A genotyping microarray revealed five regions that were retained in wasps with decreased memory retention. Independent introgressions of individual candidate regions were created using linked molecular markers and tested for memory retention. One region on chromosome 1 (spanning ∼5.8 cM) and another on chromosome 5 (spanning ∼25.6 cM) resulted in decreased memory after 72 h, without affecting 24-h-memory retention. This phenotype was observed in both heterozygous and homozygous individuals. Transcription factor CCAAT/enhancer-binding protein and a dopamine receptor, both with a known function in memory formation, are within these genomic regions and are candidates for the regulation of memory retention. Concluding, this study demonstrates a powerful approach to study variation in memory retention and provides a basis for future research on its genetic basis.

Is the salience of the distinctive features of similar stimuli affected by stimulus preexposure schedule and length?

Following intermixed or blocked preexposures to two similar stimuli (AX and BX), rats received a single taste aversion conditioning and test trial with a compound composed of the distinctive flavors of the stimuli (AB). AB consumption was lower after intermixed than blocked preexposures (Experiments 1 and 2), regardless of whether rats received few (4) or many (12) stimulus presentations during preexposure, while overall consumption of AB was not affected by the length of preexposure (Experiment 2). Furthermore, consumption of the AB compound in a control group not receiving any preexposure trial was similar to that found for the intermixed preexposure condition but lower than that found for the blocked condition (Experiment 3). Conditioning to the AB compound was always successfully established, but it seemed to be unaffected by the preexposure schedule or its length. These findings are discussed in terms of whether salience is the stimulus property being modified by the preexposure schedule.