Regional synapse gain and loss accompany memory formation in larval zebrafish.

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670-675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604-607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull 57, 397-399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

Behaviorally Conditioned Immune Responses: "To Learn New Things, Read Old Books and Papers".

BACKGROUND: More than a century ago, experimental work and clinical observations revealed the functional communication between the brain and the peripheral immune system. This is documented on the one hand by studies first demonstrating the effects of catecholamines on the circulation of leukocytes in experimental animals and humans, and on the other hand via the work of Russian physiologist Ivan Petrovic Pavlov and his coworkers, reporting observations that associative learning can modify peripheral immune functions. This work later fell into oblivion since little was known about the endocrine and immune system's function and even less about the underlying mechanisms of how learning, a central nervous system activity, could affect peripheral immune responses. SUMMARY: In this article, we embark on a fascinating exploration of the historical trajectory of behaviorally conditioned immune responses. KEY MESSAGE: We will pay homage to the visionary scientists who laid the groundwork for this field of research, tracing its evolution from early theories of how associative learning can affect immunity to the modern-day insights that behavioral conditioning of pharmacological responses can be exploited to improve the efficacy of medical interventions for patients.

The effects of parental presence on amygdala and mPFC activation during fear conditioning: An exploratory study.

Learning safe versus dangerous cues is crucial for survival. During development, parents can influence fear learning by buffering their children's stress response and increasing exploration of potentially aversive stimuli. Rodent findings suggest that these behavioral effects are mediated through parental presence modulation of the amygdala and medial prefrontal cortex (mPFC). Here, we investigated whether similar parental modulation of amygdala and mPFC during fear learning occurs in humans. Using a within-subjects design, behavioral (final N = 48, 6-17 years, mean = 11.61, SD = 2.84, 60% females/40% males) and neuroimaging data (final N = 39, 6-17 years, mean = 12.03, SD = 2.98, 59% females/41% males) were acquired during a classical fear conditioning task, which included a CS+ followed by an aversive noise (US; 75% reinforcement rate) and a CS-. Conditioning occurred once in physical contact with the participant's parent and once alone (order counterbalanced). Region of interest analyses examined the unconditioned stress response by BOLD activation to the US (vs. implicit baseline) and learning by activation to the CS+ (vs. CS-). Results showed that during US presentation, parental presence reduced the centromedial amygdala activity, suggesting buffering of the unconditioned stress response. In response to learned stimuli, parental presence reduced mPFC activity to the CS+ (relative to the CS-), although this result did not survive multiple comparisons' correction. These preliminary findings indicate that parents modulate amygdala and mPFC activity during exposure to unconditioned and conditioned fear stimuli, potentially providing insight into the neural mechanisms by which parents act as a social buffer during fear learning. RESEARCH HIGHLIGHTS: (1)This study used a within-participant experimental design to investigate how parental presence (vs. absence) affects youth's neural responses in a classical fear conditioning task. (2)Parental presence reduced the youth's centromedial amygdala activation to the unconditioned stimulus (US), suggesting parental buffering of the neural unconditioned response (UR). (3)Parental presence reduced the youth's mPFC activation to a conditioned threat cue (CS+) compared to a safety cue (CS-), suggesting possible parental modulation of fear learning.

Generalization of Costly Pain-Related Avoidance Based on Real-Life Categorical Knowledge.

Avoiding activities posing bodily threat is adaptive. However, spreading of avoidance to safe activities may cause functional disability in people with chronic pain. We investigated whether costly pain-related avoidance would generalize from one activity to another on the basis of real-life categorical knowledge in 40 pain-free people (30 female; mean age = 25 years; university students and public of Maastricht, The Netherlands). In a computer task, participants moved a joystick to complete activities from two categories (gardening and cleaning). During activities from the avoidance category, pain could be avoided at the cost of task efficiency by deviating from a short, pain-associated joystick movement. Activities from the safe category were never painful. Subsequently, we tested generalization of avoidance to novel pain-free activities from both categories. Participants generalized avoidance to novel activities from the avoidance category despite the novel activities not being paired with pain and despite avoidance costs, suggesting that costly (pain-related) avoidance generalizes from one activity to another on the basis of category knowledge and can thus be wide reaching, creating detrimental consequences.

Emulating learning behavior in a flexible device with self-formed Ag dewetted nanostructure as active element.

Neuromorphic devices are a promising alternative to the traditional von Neumann architecture. These devices have the potential to achieve high-speed, efficient, and low-power artificial intelligence. Flexibility is required in these devices so that they can bend and flex without causing damage to the underlying electronics. This feature shows a possible use in applications that require flexible electronics, such as robotics and wearable electronics. Here, we report a flexible self-formed Ag-based neuromorphic device that emulates various brain-inspired synaptic activities, such as short-term plasticity and long-term potentiation (STP and LTP) in both the flat and bent states. Half and full-integer quantum conductance jumps were also observed in the flat and bent states. The device showed excellent switching and endurance behaviors. The classical conditioning could be emulated even in the bent state.

Spatial organization of functional clusters representing reward and movement information in the striatal direct and indirect pathways.

To obtain insights into striatal neural processes underlying reward-based learning and movement control, we examined spatial organizations of striatal neurons related to movement and reward-based learning. For this, we recorded the activity of direct- and indirect-pathway neurons (D1 and A2a receptor-expressing neurons, respectively) in mice engaged in probabilistic classical conditioning and open-field free exploration. We found broadly organized functional clusters of striatal neurons in the direct as well as indirect pathways for both movement- and reward-related variables. Functional clusters for different variables were partially overlapping in both pathways, but the overlap between outcome- and value-related functional clusters was greater in the indirect than direct pathway. Also, value-related spatial clusters were progressively refined during classical conditioning. Our study shows the broad and learning-dependent spatial organization of functional clusters of dorsal striatal neurons in the direct and indirect pathways. These findings further argue against the classic model of the basal ganglia and support the importance of spatiotemporal patterns of striatal neuronal ensemble activity in the control of behavior.

From eye-blinks to state construction: Diagnostic benchmarks for online representation learning.

We present three new diagnostic prediction problems inspired by classical-conditioning experiments to facilitate research in online prediction learning. Experiments in classical conditioning show that animals such as rabbits, pigeons, and dogs can make long temporal associations that enable multi-step prediction. To replicate this remarkable ability, an agent must construct an internal state representation that summarizes its interaction history. Recurrent neural networks can automatically construct state and learn temporal associations. However, the current training methods are prohibitively expensive for online prediction-continual learning on every time step-which is the focus of this paper. Our proposed problems test the learning capabilities that animals readily exhibit and highlight the limitations of the current recurrent learning methods. While the proposed problems are nontrivial, they are still amenable to extensive testing and analysis in the small-compute regime, thereby enabling researchers to study issues in isolation, ultimately accelerating progress towards scalable online representation learning methods.

Fear Conditioning by Proxy: The Role of High Affinity Nicotinic Acetylcholine Receptors.

Observational fear-learning studies in genetically modified animals enable the investigation of the mechanisms underlying the social transmission of fear-related information. Here, we used a three-day protocol to examine fear conditioning by proxy (FCbP) in wild-type mice (C57BL/6J) and mice lacking the ß2-subunit of the nicotinic acetylcholine receptor (nAChR). Male animals of both genotypes were exposed to a previously fear-conditioned (FC) cage mate during the presentation of the conditioned stimulus (CS, tone). On the following day, observer (FCbP) mice were tested for fear reactions to the tone: none of the ß2-KO mice froze to the stimulus, while 30% of the wild-type mice expressed significant freezing. An investigation of the possible factors that predicted the fear response revealed that only wild-type mice that exhibited enhanced and more flexible social interaction with the FC cage mate during tone presentations (Day 2) expressed fear toward the CS (Day-3). Our results indicate that (i) FCbP is possible in mice; (ii) the social transmission of fear depends on the interaction pattern between animals during the FCbP session and (iii) ß2-KO mice display a more rigid interaction pattern compared to wild-type mice and are unable to acquire such information. These data suggest that ß2-nAChRs influence observational fear learning indirectly through their effect on social behaviour.

Japanese written pseudowords can be conditioned to Japanese spoken words with positive, negative, and active emotions.

This study aimed to examine whether Japanese participants condition spoken words' meanings to written pseudowords. In Survey 1, we selected spoken words associated with negative (α = .91) and positive (α = .79) features for Experiment 1 and passive (α = .90) and active (α = .80) features for Experiment 2. In Experiment 1, participants evaluated four written pseudowords' emotional valence using a 7-point semantic differential scale (1: negative; 7: positive) before and after conditioning spoken words with negative, neutral, or positive features to each pseudoword. In the conditioning phase, participants read each pseudoword, listened to a spoken word, and verbally repeated each spoken word. The results showed that a pseudoword was conditioned to spoken words with positive and negative features. In Experiment 2, participants evaluated four pseudowords' activeness using a 7-point semantic differential scale (1: passive; 7: active) before and after conditioning spoken words of passive, neutral, and active features to each written pseudoword. In the conditioning phase, the participants read each written pseudoword, listened to a spoken word, and repeated the spoken word. The results showed that the activeness evaluations were more increased for pseudowords conditioned to spoken words of active and neutral features after conditioning than before conditioning but were unchanged for a pseudoword conditioned to those with passive features before and after conditioning. Additonally, Survey 2's results showed that although the positiveness and activeness responses of the words used in Experiments 1 and 2 were controlled well, the lack of significant differences among positiveness responses of words may influence the evaluative conditioning in Experiment 2. That is, when participants condition passive (low arousal) words' activeness (arousal) ratings to those of pseudowords, words' positiveness (valence) ratings would be important in the evaluative conditioning. Our findings suggest that participants can condition spoken word meanings of preference and activeness to those of written pseudowords. It also indicates that linguistically evaluative conditioning's effects are robust in a non-alphabetic language.

[Conditioning of the immune system-Already clinically usable?] / Konditionierung des Immunsystems ­ Schon klinisch nutzbar?

The brain and the immune system permanently exchange information via various neuronal and humoral signaling pathways. This communication network forms the basis for controlling peripheral immune functions via associative learning or conditioning processes. Establishing a learned immune reaction, an immunomodulatory drug that represents the unconditioned stimulus (US) is paired with a new odor or taste stimulus. Re-presentating this previously neutral odor or taste stimulus, its now functions as a conditioned stimulus (CS) and triggers reactions in the immune system similar to those formerly induced by the drug used as US. Using different learning protocols, it was possible to condition immunopharmacological effects in animal disease models, such as lupus erythematosus, contact allergy or rheumatoid arthritis, thereby reducing disease symptoms. Preliminary experimental studies in healthy volunteers and patients confirmed a possible clinical use of learned immune responses with the aim of using associative learning protocols as complementary measures to pharmacological interventions in clinical practice in order to reduce drug doses and thus undesirable drug side effects while maintaining therapeutic efficacy. However, there is still a great need for further research to understand the mechanisms of learned immune responses in preclinical studies and to optimize the associative learning processes for using them in the clinical routine in studies with healthy volunteers and patients.

Introducing the Fear Learning and Anxiety Response (FLARe) app and web portal for the remote delivery of fear conditioning experiments.

Experimental paradigms measuring key psychological constructs can enhance our understanding of mechanisms underlying human psychological well-being and mental health. Delivering such paradigms remotely affords opportunities to reach larger, more representative samples than is typically possible with in-person research. The efficiency gained from remote delivery makes it easier to test replication of previously established effects in well-powered samples. There are several challenges to the successful development and delivery of remote experimental paradigms, including use of an appropriate delivery platform, identifying feasible outcome measures, and metrics of participant compliance. In this paper, we present FLARe (Fear Learning and Anxiety Response), open-source software in the form of a smartphone app and web portal for the creation and delivery of remote fear conditioning experiments. We describe the benefits and challenges associated with the creation of a remote delivery platform for fear conditioning, before presenting in detail the resultant software suite, and one instance of deploying this using the FLARe Research infrastructure. We provide examples of the application of FLARe to several research questions which illustrate the benefits of the remote approach to experiment delivery. The FLARe smartphone app and web portal are available for use by other researchers and have been designed to be user-friendly and intuitive. We hope that FLARe will be a useful tool for those interested in conducting well-powered fear conditioning studies to inform our understanding of the development and treatment of anxiety disorders.

A VTA GABAergic computational model of dissociated reward prediction error computation in classical conditioning.

Classical Conditioning is a fundamental learning mechanism where the Ventral Striatum is generally thought to be the source of inhibition to Ventral Tegmental Area (VTA) Dopamine neurons when a reward is expected. However, recent evidences point to a new candidate in VTA GABA encoding expectation for computing the reward prediction error in the VTA. In this system-level computational model, the VTA GABA signal is hypothesised to be a combination of magnitude and timing computed in the Peduncolopontine and Ventral Striatum respectively. This dissociation enables the model to explain recent results wherein Ventral Striatum lesions affected the temporal expectation of the reward but the magnitude of the reward was intact. This model also exhibits other features in classical conditioning namely, progressively decreasing firing for early rewards closer to the actual reward, twin peaks of VTA dopamine during training and cancellation of US dopamine after training.

Serotonin 1B receptor effects on response inhibition are independent of inhibitory learning.

Impulsivity is defined in terms of deficits in instrumental response inhibition, when the inability to withhold an action produces a negative outcome. However, there are many behavioral and cognitive constructs which theoretically could contribute to disordered impulsivity, including Pavlovian responding, which few studies have considered in this context. In the present set of studies, we examine Pavlovian inhibitory learning and excitatory responding in a mouse model for dysregulated impulsivity, specifically, mice lacking the serotonin 1B receptor (5-HT1BR). Consistent with previous results, we show that these mice display increased impulsivity as measured by premature responding in the operant 5-choice serial reaction time test. In a Pavlovian conditioned inhibition paradigm, they also show a decreased ability to withhold responding, but importantly have an intact ability to learn inhibitory associations. In a Pavlovian appetitive conditioning experiment, 5-HT1BR knockout mice show normal responding under a positive contingency schedule, however, they display increased responding to cues presented on an independent schedule from reinforcement in a zero contingency schedule. Interestingly this difference does not occur when the cues are explicitly unpaired in a negative contingency schedule, nor during a 25% reinforcement schedule. Overall, while our results show that the deficits in operant response inhibition in mice lacking 5-HT1BR are likely not due to Pavlovian inhibitory or excitatory learning, it is relevant to consider associative learning in the context of dysregulated impulsive behavior.

Learning to anticipate mate presence shapes individual sex roles in the hermaphroditic pond snail, Lymnaea stagnalis.

Despite being simultaneously male and female, hermaphrodites may still need to assume the male or female sexual role in a mating encounter, with the option to swap roles afterwards. For the great pond snail, Lymnaea stagnalis, deciding which sexual role to perform has important consequences, since sperm transfer and male reproductive success can be decreased. We hypothesised that detecting cues that indicate a possible mating encounter could help them to adapt their mating behaviour. Therefore, we experimentally assessed whether signalling the presence of a conspecific with an odour can affect the sexual role of Lymnaea stagnalis. The results showed that learning resulted in either an increased ability to mate as a male or in faster mating compared to the control group. These findings reveal that learning shapes the mating dynamics of Lymnaea stagnalis, thus showing that cognitive processes not only affect mating in separate-sexed species but also in hermaphrodites.

Classically conditioned modulation of pain depends on stimulus intensity.

Innocuous cues that become associated with pain can enhance pain. This is termed classically conditioned hyperalgesia. The size of this effect varies under different conditions. We aimed to test whether the sensitising effect of pain-associated cues depends on the intensity of the paired test stimulus. To do this, two virtual reality environments were paired with either painful or non-painful vibrotactile stimuli in a counterbalanced fashion. The differential effect of the two environments was evaluated using pain intensity ratings of paired electrocutaneous test stimuli at three different intensity levels. Forty healthy participants were included in the study; 30 participants experienced sufficient pain during the learning phase and were included in the main analysis. An effect of environment (p = 0.014) and interaction between environment and test stimulus intensity was found (p = 0.046). Only the most intense test stimulus was modulated by environment. While the effect was small, the results are consistent with the proposition that pain-associated cues may induce hyperalgesia to some degree, under certain conditions. In particular, results highlight the potential relevance of stimulus intensity during and after the initial painful experience. Further attention is needed to comprehensively understand the variables that impact classically conditioned hyperalgesia.

Cortical Processing of Multimodal Sensory Learning in Human Neonates.

Following birth, infants must immediately process and rapidly adapt to the array of unknown sensory experiences associated with their new ex-utero environment. However, although it is known that unimodal stimuli induce activity in the corresponding primary sensory cortices of the newborn brain, it is unclear how multimodal stimuli are processed and integrated across modalities. The latter is essential for learning and understanding environmental contingencies through encoding relationships between sensory experiences; and ultimately likely subserves development of life-long skills such as speech and language. Here, for the first time, we map the intracerebral processing which underlies auditory-sensorimotor classical conditioning in a group of 13 neonates (median gestational age at birth: 38 weeks + 4 days, range: 32 weeks + 2 days to 41 weeks + 6 days; median postmenstrual age at scan: 40 weeks + 5 days, range: 38 weeks + 3 days to 42 weeks + 1 days) with blood-oxygen-level-dependent (BOLD) functional magnetic resonance imaging (MRI) and magnetic resonance (MR) compatible robotics. We demonstrate that classical conditioning can induce crossmodal changes within putative unimodal sensory cortex even in the absence of its archetypal substrate. Our results also suggest that multimodal learning is associated with network wide activity within the conditioned neural system. These findings suggest that in early life, external multimodal sensory stimulation and integration shapes activity in the developing cortex and may influence its associated functional network architecture.

Decrease of glycogen synthase kinase 3ß phosphorylation in the rat nucleus accumbens shell is necessary for amphetamineinduced conditioned locomotor activity.

Phosphorylation levels of glycogen synthase kinase 3ß (GSK3ß) negatively correlated with psychomotor stimulant-induced locomotor activity. Locomotor sensitization induced by psychomotor stimulants was previously shown to selectively accompany the decrease of GSK3ß phosphorylation in the nucleus accumbens (NAcc) core, suggesting that intact GSK3ß activity in this region is necessary for psychomotor stimulants to produce locomotor sensitization. Similarly, GSK3ß in the NAcc was also implicated in mediating the conditioned effects formed by the associations of psychomotor stimulants. However, it remains undetermined whether GSK3ß plays a differential role in the two sub-regions (core and shell) of the NAcc in the expression of drug-conditioned behaviors. In the present study, we found that GSK3ß phosphorylation was significantly lower in the NAcc shell obtained from rats expressing amphetamine (AMPH)-induced conditioned locomotor activity. Further, we demonstrated that these effects were normalized by treatment with lithium chloride, a GSK3ß inhibitor. These results suggest that the behavior produced by AMPH itself and a conditioned behavior formed by associations with AMPH are differentially mediated by the two sub-regions of the NAcc.

Sleep and conditioning of the siphon withdrawal reflex in Aplysia.

Sleep is essential for memory consolidation after learning as shown in mammals and invertebrates such as bees and flies. Aplysia californica displays sleep, and sleep in this mollusk was also found to support memory for an operant conditioning task. Here, we investigated whether sleep in Aplysia is also required for memory consolidation in a simpler type of learning, i.e. the conditioning of the siphon withdrawal reflex. Two groups of animals (Wake, Sleep, each n=11) were conditioned on the siphon withdrawal reflex, with the training following a classical conditioning procedure where an electrical tail shock served as the unconditioned stimulus (US) and a tactile stimulus to the siphon as the conditioned stimulus (CS). Responses to the CS were tested before (pre-test), and 24 and 48 h after training. While Wake animals remained awake for 6 h after training, Sleep animals had undisturbed sleep. The 24 h test in both groups was combined with extinction training, i.e. the extended presentation of the CS alone over two blocks. At the 24 h test, siphon withdrawal duration in response to the CS was distinctly enhanced in both Sleep and Wake groups with no significant difference between groups, consistent with the view that consolidation of a simple conditioned reflex response does not require post-training sleep. Surprisingly, extinction training did not reverse the enhancement of responses to the CS. On the contrary, at the 48 h test, withdrawal duration in response to the CS was even further enhanced across both groups. This suggests that processes of sensitization, an even simpler non-associative type of learning, contributed to the withdrawal responses. Our study provides evidence for the hypothesis that sleep preferentially benefits consolidation of more complex learning paradigms than conditioning of simple reflexes.

Learning a non-neutral conditioned stimulus: place preference in the crab Neohelice granulata.

In the wild, being able to recognize and remember specific locations related to food sources and the associated attributes of landmarks is a cognitive trait important for survival. In the present work, we show that the crab Neohelice granulata can be trained to associate a specific environment with an appetitive reward in a conditioned place preference task. After a single training trial, when the crabs were presented with a food pellet in the target quadrant of the training arena, they were able to form a long-term memory related to the event. This memory was evident at least 24 h after training and was protein synthesis dependent. Importantly, the target area of the arena proved to be a non-neutral environment, given that animals initially avoided the target quadrant. In the present work, we introduce for the first time an associative one-trial memory paradigm including a conditioned stimulus with a clear valence performed in a crustacean.

Stimulus-dependent learning and memory in the neotropical ant Ectatomma ruidum.

Learning and memory are major cognitive processes strongly tied to the life histories of animals. In ants, chemotactile information generally plays a central role in social interaction, navigation and resource exploitation. However, in hunters, visual information should take special relevance during foraging, thus leading to differential use of information from different sensory modalities. Here, we aimed to test whether a hunter, the neotropical ant Ectatomma ruidum, differentially learns stimuli acquired through multiple sensory channels. We evaluated the performance of E. ruidum workers when trained using olfactory, mechanical, chemotactile and visual stimuli under a restrained protocol of appetitive learning. Conditioning of the maxilla labium extension response enabled control of the stimuli provided. Our results show that ants learn faster and remember for longer when trained using chemotactile or visual stimuli than when trained using olfactory and mechanical stimuli separately. These results agree with the life history of E. ruidum, characterized by a high relevance of chemotactile information acquired through antennation as well as the role of vision during hunting.