Distinct Dopamine Receptor Pathways Underlie the Temporal Sensitivity of Associative Learning.

Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. Here, we show that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, we show that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. Our results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment.

The Drosophila Mushroom Body: From Architecture to Algorithm in a Learning Circuit.

The Drosophila brain contains a relatively simple circuit for forming Pavlovian associations, yet it achieves many operations common across memory systems. Recent advances have established a clear framework for Drosophila learning and revealed the following key operations: a) pattern separation, whereby dense combinatorial representations of odors are preprocessed to generate highly specific, nonoverlapping odor patterns used for learning; b) convergence, in which sensory information is funneled to a small set of output neurons that guide behavioral actions; c) plasticity, where changing the mapping of sensory input to behavioral output requires a strong reinforcement signal, which is also modulated by internal state and environmental context; and d) modularization, in which a memory consists of multiple parallel traces, which are distinct in stability and flexibility and exist in anatomically well-defined modules within the network. Cross-module interactions allow for higher-order effects where past experience influences future learning. Many of these operations have parallels with processes of memory formation and action selection in more complex brains.

Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation.

Microtubule-associated protein tau is a central factor in Alzheimer's disease and other tauopathies. However, the physiological functions of tau are unclear. Here, we used proximity-labelling proteomics to chart tau interactomes in primary neurons and mouse brains in vivo. Tau interactors map onto pathways of cytoskeletal, synaptic vesicle and postsynaptic receptor regulation and show significant enrichment for Parkinson's, Alzheimer's and prion disease. We find that tau interacts with and dose-dependently reduces the activity of N-ethylmaleimide sensitive fusion protein (NSF), a vesicular ATPase essential for AMPA-type glutamate receptor (AMPAR) trafficking. Tau-deficient (tau-/- ) neurons showed mislocalised expression of NSF and enhanced synaptic AMPAR surface levels, reversible through the expression of human tau or inhibition of NSF. Consequently, enhanced AMPAR-mediated associative and object recognition memory in tau-/- mice is suppressed by both hippocampal tau and infusion with an NSF-inhibiting peptide. Pathologic mutant tau from mouse models or Alzheimer's disease significantly enhances NSF inhibition. Our results map neuronal tau interactomes and delineate a functional link of tau with NSF in plasticity-associated AMPAR-trafficking and memory.

Associative learning in the cnidarian Nematostella vectensis.

The ability to learn and form memories allows animals to adapt their behavior based on previous experiences. Associative learning, the process through which organisms learn about the relationship between two distinct events, has been extensively studied in various animal taxa. However, the existence of associative learning, prior to the emergence of centralized nervous systems in bilaterian animals, remains unclear. Cnidarians such as sea anemones or jellyfish possess a nerve net, which lacks centralization. As the sister group to bilaterians, they are particularly well suited for studying the evolution of nervous system functions. Here, we probe the capacity of the starlet sea anemone Nematostella vectensis to form associative memories by using a classical conditioning approach. We developed a protocol combining light as the conditioned stimulus with an electric shock as the aversive unconditioned stimulus. After repetitive training, animals exhibited a conditioned response to light alone-indicating that they learned the association. In contrast, all control conditions did not form associative memories. Besides shedding light on an aspect of cnidarian behavior, these results root associative learning before the emergence of NS centralization in the metazoan lineage and raise fundamental questions about the origin and evolution of cognition in brainless animals.

Unconscious Manipulation of Conceptual Representations with Decoded Neurofeedback Impacts Search Behavior.

The necessity of conscious awareness in human learning has been a long-standing topic in psychology and neuroscience. Previous research on non-conscious associative learning is limited by the low signal-to-noise ratio of the subliminal stimulus, and the evidence remains controversial, including failures to replicate. Using functional MRI decoded neurofeedback, we guided participants from both sexes to generate neural patterns akin to those observed when visually perceiving real-world entities (e.g., dogs). Importantly, participants remained unaware of the actual content represented by these patterns. We utilized an associative DecNef approach to imbue perceptual meaning (e.g., dogs) into Japanese hiragana characters that held no inherent meaning for our participants, bypassing a conscious link between the characters and the dogs concept. Despite their lack of awareness regarding the neurofeedback objective, participants successfully learned to activate the target perceptual representations in the bilateral fusiform. The behavioral significance of our training was evaluated in a visual search task. DecNef and control participants searched for dogs or scissors targets that were pre-cued by the hiragana used during DecNef training or by a control hiragana. The DecNef hiragana did not prime search for its associated target but, strikingly, participants were impaired at searching for the targeted perceptual category. Hence, conscious awareness may function to support higher-order associative learning. Meanwhile, lower-level forms of re-learning, modification, or plasticity in existing neural representations can occur unconsciously, with behavioral consequences outside the original training context. The work also provides an account of DecNef effects in terms of neural representational drift.

Long-Horizon Associative Learning Explains Human Sensitivity to Statistical and Network Structures in Auditory Sequences.

Networks are a useful mathematical tool for capturing the complexity of the world. In a previous behavioral study, we showed that human adults were sensitive to the high-level network structure underlying auditory sequences, even when presented with incomplete information. Their performance was best explained by a mathematical model compatible with associative learning principles, based on the integration of the transition probabilities between adjacent and nonadjacent elements with a memory decay. In the present study, we explored the neural correlates of this hypothesis via magnetoencephalography (MEG). Participants (N = 23, 16 females) passively listened to sequences of tones organized in a sparse community network structure comprising two communities. An early difference (∼150 ms) was observed in the brain responses to tone transitions with similar transition probability but occurring either within or between communities. This result implies a rapid and automatic encoding of the sequence structure. Using time-resolved decoding, we estimated the duration and overlap of the representation of each tone. The decoding performance exhibited exponential decay, resulting in a significant overlap between the representations of successive tones. Based on this extended decay profile, we estimated a long-horizon associative learning novelty index for each transition and found a correlation of this measure with the MEG signal. Overall, our study sheds light on the neural mechanisms underlying human sensitivity to network structures and highlights the potential role of Hebbian-like mechanisms in supporting learning at various temporal scales.

Pupillary Responses Reflect Dynamic Changes in Multiple Cognitive Factors During Associative Learning in Primates.

Associative learning involves complex interactions of multiple cognitive factors. While adult subjects can articulate these factors verbally, for model animals such as macaques, we rely on behavioral outputs. In our study, we used pupillary responses as an alternative measure to capture these underlying cognitive changes. We recorded the dynamic changes in the pupils of three male macaques when they learned the associations between visual stimuli and reward sizes under the classical Pavlovian experimental paradigm. We found that during the long-term learning process, the gradual changes in the pupillary response reflect the changes in the cognitive state of the animals. The pupillary response can be explained by a linear combination of components corresponding to multiple cognitive factors. These components reflect the impact of visual stimuli on the pupils, the prediction of reward values associated with the visual stimuli, and the macaques' understanding of the current experimental reward rules. The changing patterns of these factors during interday and intraday learning clearly demonstrate the enhancement of current reward-stimulus association and the weakening of previous reward-stimulus association. Our study shows that the dynamic response of pupils can serve as an objective indicator to characterize the psychological changes of animals, understand their learning process, and provide important tools for exploring animal behavior during the learning process.

Causal Influence of Linguistic Learning on Perceptual and Conceptual Processing: A Brain-Constrained Deep Neural Network Study of Proper Names and Category Terms.

Language influences cognitive and conceptual processing, but the mechanisms through which such causal effects are realized in the human brain remain unknown. Here, we use a brain-constrained deep neural network model of category formation and symbol learning and analyze the emergent model's internal mechanisms at the neural circuit level. In one set of simulations, the network was presented with similar patterns of neural activity indexing instances of objects and actions belonging to the same categories. Biologically realistic Hebbian learning led to the formation of instance-specific neurons distributed across multiple areas of the network, and, in addition, to cell assembly circuits of "shared" neurons responding to all category instances-the network correlates of conceptual categories. In two separate sets of simulations, the network learned the same patterns together with symbols for individual instances ["proper names" (PN)] or symbols related to classes of instances sharing common features ["category terms" (CT)]. Learning CT remarkably increased the number of shared neurons in the network, thereby making category representations more robust while reducing the number of neurons of instance-specific ones. In contrast, proper name learning prevented a substantial reduction of instance-specific neurons and blocked the overgrowth of category general cells. Representational similarity analysis further confirmed that the neural activity patterns of category instances became more similar to each other after category-term learning, relative to both learning with PN and without any symbols. These network-based mechanisms for concepts, PN, and CT explain why and how symbol learning changes object perception and memory, as revealed by experimental studies.

Neural correlates of retrospective memory confidence during face-name associative learning.

The ability to accurately assess one's own memory performance during learning is essential for adaptive behavior, but the brain mechanisms underlying this metamemory function are not well understood. We investigated the neural correlates of memory accuracy and retrospective memory confidence in a face-name associative learning task using magnetoencephalography in healthy young adults (n = 32). We found that high retrospective confidence was associated with stronger occipital event-related fields during encoding and widespread event-related fields during retrieval compared to low confidence. On the other hand, memory accuracy was linked to medial temporal activities during both encoding and retrieval, but only in low-confidence trials. A decrease in oscillatory power at alpha/beta bands in the parietal regions during retrieval was associated with higher memory confidence. In addition, representational similarity analysis at the single-trial level revealed distributed but differentiable neural activities associated with memory accuracy and confidence during both encoding and retrieval. In summary, our study unveiled distinct neural activity patterns related to memory confidence and accuracy during associative learning and underscored the crucial role of parietal regions in metamemory.

Cyclic nucleotide-induced bidirectional long-term synaptic plasticity in Drosophila mushroom body.

Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odour-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. Here, we developed a system to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrated that they are indeed an exception in which activation of the cAMP-protein kinase A pathway induces LTD. Contrary to the prevailing model, our cAMP imaging found no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, we found that forskolin-induced cAMP increase alone was insufficient for plasticity induction; it additionally required simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induced slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioural study. Finally, KC subtype-specific interrogation of synapses revealed that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, our work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model. KEY POINTS: Although presynaptic cAMP increase generally facilitates synapses, olfactory associative learning in Drosophila, which depends on dopamine and cAMP signalling genes, induces long-term depression (LTD) at the mushroom body output synapses. By combining electrophysiology, pharmacology and optogenetics, we directly demonstrate that these synapses are an exception where activation of the cAMP-protein kinase A pathway leads to presynaptic LTD. Dopamine- or forskolin-induced cAMP increase alone is not sufficient for LTD induction; neuronal activity, which has been believed to trigger cAMP synthesis in synergy with dopamine input, is required in the downstream pathway of cAMP. In contrast to cAMP, activation of the cGMP pathway paired with neuronal activity induces presynaptic long-term potentiation, which explains behaviourally observed opposing actions of transmitters co-released by dopaminergic neurons. Our work not only revises the role of cAMP in synaptic plasticity, but also provides essential methods to address physiological mechanisms of synaptic plasticity in this important model system.

Contextual fear conditioning in zebrafish: Influence of different shock frequencies, context, and pharmacological modulation on behavior.

Contextual fear conditioning is a protocol used to assess associative learning across species, including fish. Here, our goal was to expand the analysis of behavioral parameters that may reflect aversive behaviors in a contextual fear conditioning protocol using adult zebrafish (Danio rerio) and to verify how such parameters can be modulated. First, we analyzed the influence of an aversive stimulus (3 mild electric shocks for 5 s each at frequencies of 10, 100 or 1000 Hz) on fish behavior, and their ability to elicit fear responses in the absence of shock during a test session. To confirm whether the aversive responses are context-dependent, behaviors were also measured in a different experimental environment in a test session. Furthermore, we investigated the effects of dizocilpine (MK-801, 2 mg/kg, i.p.) on fear-related responses. Zebrafish showed significant changes in baseline activity immediately after shock exposure in the training session, in which 100 Hz induced robust contextual fear responses during the test session. Importantly, when introduced to a different environment, animals exposed to the aversive stimulus did not show any differences in locomotion and immobility-related parameters. MK-801 administered after the training session reduced fear responses during the test, indicating that glutamate NMDA-receptors play a key role in the consolidation of contextual fear-related memory in zebrafish. In conclusion, by further exploring fear-related behaviors in a contextual fear conditioning task, we show the effects of different shock frequencies and confirm the importance of context on aversive responses for associative learning in zebrafish. Additionally, our data support the use of zebrafish in contextual fear conditioning tasks, as well as for advancing pharmacological studies related to associative learning in translational neurobehavioral research.

Habituation to a predatory stimulus in a harvester (Arachnida, Opiliones).

Several studies have investigated habituation in a defensive context, but few have addressed responses to dangerous stimuli. In such cases, animals should not habituate since this could cost their lives. Here we have stimulated individuals of the harvester Mischonyx squalidus with a predatory stimulus (squeezing with tweezers) in repeated trials within and between days, and measured the occurrence and magnitude of nipping, a defensive behavior. Contrary to our expectations, they did habituate to this stimulus. The probability and magnitude of response declined over trials during each of three days of testing in a typical habituation pattern. During the trials we also observed other defensive behaviors. We discuss our results mainly considering alternative defensive responses. Our data show that we lack information on (1) the role played by the ambiguity of stimuli, (2) the role played by subsequent stimuli and (3) the importance of the array of defensive behaviors of a species in understanding habituation. Although ubiquitous across animals and therefore expected, habituation is described for the first time in the order Opiliones.

Chunking as a function of sequence length.

Chunking mechanisms are central to several cognitive processes. During the acquisition of visuo-motor sequences, it is commonly reported that these sequences are segmented into chunks leading to more fluid, rapid, and accurate performances. The question of a chunk's storage capacity has been often investigated but little is known about the dynamics of chunk size evolution relative to sequence length. In two experiments, we studied the dynamics and the evolution of a sequence's chunking pattern as a function of sequence length in a non-human primate species (Guinea baboons, Papio papio). Using an operant conditioning device, baboons had to point on a touch screen to a moving target. In Experiment 1, they had to produce repeatedly the same sequence of 4 movements during 2000 trials. In Experiment 2, the sequence was composed of 5 movements and was repeated 4000 times. For both lengths, baboons initially produced small chunks that became fewer and longer with practice. Moreover, the dynamics and the evolution of the chunking pattern varied as a function of sequence length. Finally, with extended practice (i.e., more than 2000 trials), we observed that the mean chunk size reached a plateau indicating that there are fundamental limits to chunking processes that also depend on sequence length. These data therefore provide new empirical evidence for understanding the general properties of chunking mechanisms in sequence learning.

Multiple mechanisms of visual prediction as revealed by the timecourse of scene-object facilitation.

Not only semantic, but also recently learned arbitrary associations have the potential to facilitate visual processing in everyday life-for example, knowledge of a (moveable) object's location at a specific time may facilitate visual processing of that object. In our prior work, we showed that previewing a scene can facilitate processing of recently associated objects at the level of visual analysis (Smith and Federmeier in Journal of Cognitive Neuroscience, 32(5):783-803, 2020). In the current study, we assess how rapidly this facilitation unfolds by manipulating scene preview duration. We then compare our results to studies using well-learned object-scene associations in a first-pass assessment of whether systems consolidation might speed up high-level visual prediction. In two ERP experiments (N = 60), we had participants study categorically organized novel object-scene pairs in an explicit paired associate learning task. At test, we varied contextual pre-exposure duration, both between (200 vs. 2500 ms) and within subjects (0-2500 ms). We examined the N300, an event-related potential component linked to high-level visual processing of objects and scenes and found that N300 effects of scene congruity increase with longer scene previews, up to approximately 1-2 s. Similar results were obtained for response times and in a separate component-neutral ERP analysis of visual template matching. Our findings contrast with prior evidence that scenes can rapidly facilitate visual processing of commonly associated objects. This raises the possibility that systems consolidation might mediate different kinds of predictive processing with different temporal profiles.

Don't throw the associative baby out with the Bayesian bathwater: Children are more associative when reasoning retrospectively under information processing demands.

Causal reasoning is a fundamental cognitive ability that enables individuals to learn about the complex interactions in the world around them. However, the mechanisms that underpin causal reasoning are not well understood. For example, it remains unresolved whether children's causal inferences are best explained by Bayesian inference or associative learning. The two experiments and computational models reported here were designed to examine whether 5- and 6-year-olds will retrospectively reevaluate objects-that is, adjust their beliefs about the causal status of some objects presented at an earlier point in time based on the observed causal status of other objects presented at a later point in time-when asked to reason about 3 and 4 objects and under varying degrees of information processing demands. Additionally, the experiments and models were designed to determine whether children's retrospective reevaluations were best explained by associative learning, Bayesian inference, or some combination of both. The results indicated that participants retrospectively reevaluated causal inferences under minimal information-processing demands (Experiment 1) but failed to do so under greater information processing demands (Experiment 2) and that their performance was better captured by an associative learning mechanism, with less support for descriptions that rely on Bayesian inference. RESEARCH HIGHLIGHTS: Five- and 6-year-old children engage in retrospective reevaluation under minimal information-processing demands (Experiment 1). Five- and 6-year-old children do not engage in retrospective reevaluation under more extensive information-processing demands (Experiment 2). Across both experiments, children's retrospective reevaluations were better explained by a simple associative learning model, with only minimal support for a simple Bayesian model. These data contribute to our understanding of the cognitive mechanisms by which children make causal judgements.

Second-order fear conditioning involves formation of competing stimulus-danger and stimulus-safety associations.

How do animals process experiences that provide contradictory information? The present study addressed this question using second-order fear conditioning in rats. In second-order conditioning, rats are conditioned to fear a stimulus, S1, through its pairings with foot-shock (stage 1); and some days later, a second stimulus, S2, through its pairings with the already-conditioned S1 (stage 2). However, as foot-shock is never presented during conditioning to S2, we hypothesized that S2 simultaneously encodes 2 contradictory associations: one that drives fear to S2 (S2-danger) and another that reflects the absence of the expected unconditioned stimulus and partially masks that fear (e.g. S2-safety). We tested this hypothesis by manipulating the substrates of danger and safety learning in the brain (using a chemogenetic approach) and assessing the consequences for second-order fear to S2. Critically, silencing activity in the basolateral amygdala (important for danger learning) reduced fear to S2, whereas silencing activity in the infralimbic cortex (important for safety learning) enhanced fear to S2. These bidirectional changes are consistent with our hypothesis that second-order fear conditioning involves the formation of competing S2-danger and S2-safety associations. More generally, they show that a single set of experiences can produce contradictory associations and that the brain resolves the contradiction by encoding these associations in distinct brain regions.

Testing the role of associative learning in evidence-based treatments for anorexia nervosa.

Treatments for anorexia nervosa (AN) remain ineffective for many patients. Processes that can account for differential treatment outcomes remain mostly unknown. We propose that the field test the role of associative learning in current psychological treatments. We hold that this line of research could yield actionable information for understanding non-response and improving long-term outcomes. To make this argument, we define associative learning and outline its proposed role in understanding psychiatric disorders and their treatment. We then briefly review data exploring associative learning in AN. We argue that associative learning processes are implicitly implicated in existing treatments; by this rationale, baseline differences in learning may interfere with treatment response. Finally, we outline future research to test our hypotheses. Altogether, future research aimed at better understanding how associative learning may contribute to AN symptom persistence has the potential to inform novel directions in intervention research. PUBLIC SIGNIFICANCE: There is a pressing need to improve outcomes in treatments for anorexia nervosa (AN). We propose that individual differences in associative learning-the ability to form and update associations between cues, contexts, behaviors, and outcomes-may account for differential response to existing treatments. Undertaking this research could provide an understanding of how current treatments work and inform new approaches for those who may be at risk of poor outcomes.

Going back to "basics": Harlow's learning set task with wolves and dogs.

To survive and reproduce, animals need to behave adaptively by adjusting their behavior to their environment, with learning facilitating some of these processes. Dogs have become a go-to model species in comparative cognition studies, making our understanding of their learning skills paramount at multiple levels, not only with regards to basic research on their cognitive skills and the effects of domestication, but also with applied purposes such as training. In order to tackle these issues, we tested similarly raised wolves and dogs in a serial learning task inspired by Harlow's "learning set." In Phase 1, different pairs of objects were presented to the animals, one of which was baited while the other was not. Both species' performance gradually improved with each new set of objects, showing that they "learnt to learn," but no differences were found between the species in their learning speed. In Phase 2, once subjects had learned the association between one of the objects and the food reward, the contingencies were reversed and the previously unrewarded object of the same pair was now rewarded. Dogs' performance in this task seemed to be better than wolves', albeit only when considering just the first session of each reversal, suggesting that the dogs might be more flexible than wolves. Further research (possibly with the aid of refined methods such as computer-based tasks) would help ascertain whether these differences between wolves and dogs are persistent across different learning tasks.

Overcoming age differences in memory retrieval by reducing stereotype threat.

Very little is known about whether and how socioemotional factors influence age differences in associative memory. Here, we tested the hypothesis that reducing the threat induced by age-based stereotypes can reduce age differences in learning performance and strategy. Using an associative learning task, we replicated the classic finding of age differences under a high-threat condition: older adults had longer reaction times than younger adults and were much more reluctant to use memory retrieval. However, age differences were greatly diminished under a low-threat condition. These findings demonstrate that memory retrieval is an ability not entirely lost as individuals age because merely reducing stereotype threat helped restoring it. We conclude that socioemotional factors, such as stereotype threat, should be considered when evaluating younger and older adults' memory performance.

Learned switch readiness via concurrent activation of task sets: Evidence from task specificity and memory load.

Cognitive flexibility increases when switch demands increase. In task switching experiments, repeated pairing of flexibility-demanding situations with specific contexts leads subjects to become more prepared to adapt to changing task demands in those contexts. One form of such upregulated cognitive flexibility has been demonstrated with a list-wide switch probability (LWSP) effect, where switch costs are smaller in lists with frequent switches than in lists with rare switches. According to a recent proposal, the LWSP effect is supported by a concurrent activation mechanism whereby both task rules are kept available simultaneously in working memory. We conducted four experiments to test two key features in this concurrent activation account of LWSP effects. First, we asked whether the LWSP effects are limited to only the trained tasks, and second, we asked whether concurrent working memory load would reduce the LWSP effects. In Experiment 1, we replicated and extended previous findings that the LWSP manipulation modulates both performance (switch costs) and voluntary switch rates, indicating that context-driven increases in flexibility are generalizable so long as the task-sets remain the same. Results of Experiments 2 and 3 showed that novel tasks do not benefit from the concurrent activation of the two other tasks, suggesting that the LWSP effect is task specific. Experiment 4 showed that holding additional information in working memory reduces the LWSP effect. While these findings support the hypothesis of concurrent activation underlying the increased flexibility in the LWSP effect, caveats remain; additional research is needed to further test this account.