Spaced conditioned stimulus presentation facilitates the extinction of strong fear memory in mice.

Inducing fear memory extinction by re-presenting a conditioned stimulus (CS) is the foundation of exposure therapy for post-traumatic stress disorder (PTSD). Investigating differences in the ability of different CS presentation patterns to induce extinction learning is crucial for improving this type of therapy. Using a trace fear conditioning paradigm in mice, we demonstrate that spaced presentation of the CS facilitated the extinction of a strong fear memory to a greater extent than continuous CS presentation. These results lay the groundwork for developing more effective exposure therapy techniques for PTSD.

Acan downregulation in parvalbumin GABAergic cells reduces spontaneous recovery of fear memories.

While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV+) cells, restricts plasticity in the adult brain, thus reducing PV+ cell maturation could promote the suppression of fear memories following extinction training in adults. Epigenetic modifications such as histone acetylation control gene accessibility for transcription and help couple synaptic activity to changes in gene expression. Histone deacetylase 2 (Hdac2), in particular, restrains both structural and functional synaptic plasticity. However, whether and how Hdac2 controls the maturation of postnatal PV+ cells is not well understood. Here, we show that PV+- cell specific Hdac2 deletion limits spontaneous fear memory recovery in adult mice, while enhancing PV+ cell bouton remodeling and reducing perineuronal net aggregation around PV+ cells in prefrontal cortex and basolateral amygdala. Prefrontal cortex PV+ cells lacking Hdac2, show reduced expression of Acan, a critical perineuronal net component, which is rescued by Hdac2 re-expression. Pharmacological inhibition of Hdac2 before extinction training is sufficient to reduce both spontaneous fear memory recovery and Acan expression in wild-type adult mice, while these effects are occluded in PV+-cell specific Hdac2 conditional knockout mice. Finally, a brief knock-down of Acan expression mediated by intravenous siRNA delivery before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear recovery in wild-type mice. Altogether, these data suggest that controlled manipulation of PV+ cells by targeting Hdac2 activity, or the expression of its downstream effector Acan, promotes the long-term efficacy of extinction training in adults.

Conditioned preferences: Gated by experience, context, and endocrine systems.

Central to the navigation of an ever-changing environment is the ability to form positive associations with places and conspecifics. The functions of location and social conditioned preferences are often studied independently, limiting our understanding of their interplay. Furthermore, a de-emphasis on natural functions of conditioned preferences has led to neurobiological interpretations separated from ecological context. By adopting a naturalistic and ethological perspective, we uncover complexities underlying the expression of conditioned preferences. Development of conditioned preferences is a combination of motivation, reward, associative learning, and context, including for social and spatial environments. Both social- and location-dependent reward-responsive behaviors and their conditioning rely on internal state-gating mechanisms that include neuroendocrine and hormone systems such as opioids, dopamine, testosterone, estradiol, and oxytocin. Such reinforced behavior emerges from mechanisms integrating past experience and current social and environmental conditions. Moreover, social context, environmental stimuli, and internal state gate and modulate motivation and learning via associative reward, shaping the conditioning process. We highlight research incorporating these concepts, focusing on the integration of social neuroendocrine mechanisms and behavioral conditioning. We explore three paradigms: 1) conditioned place preference, 2) conditioned social preference, and 3) social conditioned place preference. We highlight nonclassical species to emphasize the naturalistic applications of these conditioned preferences. To fully appreciate the complex integration of spatial and social information, future research must identify neural networks where endocrine systems exert influence on such behaviors. Such research promises to provide valuable insights into conditioned preferences within a broader naturalistic context.

Reward behaviour is regulated by the strength of hippocampus-nucleus accumbens synapses.

Reward drives motivated behaviours and is essential for survival, and therefore there is strong evolutionary pressure to retain contextual information about rewarding stimuli. This drive may be abnormally strong, such as in addiction, or weak, such as in depression, in which anhedonia (loss of pleasure in response to rewarding stimuli) is a prominent symptom. Hippocampal input to the shell of the nucleus accumbens (NAc) is important for driving NAc activity1,2 and activity-dependent modulation of the strength of this input may contribute to the proper regulation of goal-directed behaviours. However, there have been few robust descriptions of the mechanisms that underlie the induction or expression of long-term potentiation (LTP) at these synapses, and there is, to our knowledge, no evidence about whether such plasticity contributes to reward-related behaviour. Here we show that high-frequency activity induces LTP at hippocampus-NAc synapses in mice via canonical, but dopamine-independent, mechanisms. The induction of LTP at this synapse in vivo drives conditioned place preference, and activity at this synapse is required for conditioned place preference in response to a natural reward. Conversely, chronic stress, which induces anhedonia, decreases the strength of this synapse and impairs LTP, whereas antidepressant treatment is accompanied by a reversal of these stress-induced changes. We conclude that hippocampus-NAc synapses show activity-dependent plasticity and suggest that their strength may be critical for contextual reward behaviour.

Two social minds in one brain? error-related negativity provides evidence for parallel processing pathways during social evaluation.

Several authors assume that evaluative conditioning (EC) relies on high-level propositional thinking. In contrast, the dual-process perspective proposes two processing pathways, one associative and the other propositional, contributing to EC. Dual-process theorists argue that attitudinal ambiguity resulting from these two pathways' conflicting evaluations demonstrate the involvement of both automatic and controlled processes in EC. Previously, we suggested that amplitude variations of error-related negativity and error-positivity, two well-researched event-related potentials of performance monitoring, allow for the detection of attitudinal ambiguity at the neural level. The present study utilises self-reported evaluation, categorisation performance, and neural correlates of performance monitoring to explore associative-propositional ambiguity during social attitude formation. Our results show that compared to associative-propositional harmony, attitudinal ambiguity correlates with more neutral subjective evaluations, longer response times, increased error commission, and diminished error-related negativity amplitudes. While our findings align with dual-process models, we aim to offer a propositional interpretation. We discuss dual-process theories in the context of evolutionary psychology, suggesting that associative processes may only represent a small piece of the EC puzzle.

Social buffering reduces fear expression in Wistar rats when tested in pairs, but not when retested alone.

Social buffering is a phenomenon in which the stress response of an individual can be reduced by the presence of another individual. However, little is known about the effect of social buffering on aversive after memory extinction, especially when animals are tested alone afterwards. The aim of this study was to verify the social buffering effect in rats during the extinction session of the contextual fear conditioning model and the fear response when animals are tested alone in the following day. Animals were divided into subjects and associates, with the subjects undergoing the fear conditioning protocol and the associates paired with the subjects during the fear extinction session. Across five different experiments, we tested moderate and high intensity contextual fear conditioning protocols, as well four variations of pairs: (i) two conditioned subjects, (ii) a conditioned subject and a non-conditioned associate, (iii) a conditioned subject and an associate who observed the conditioning of the partner and (iv) two conditioned subjects, with one treated with diazepam. The social buffering effect was found efficient to reduce the fear memory expression during the fear extinction session. In the moderate intensity protocol, the reduction in freezing time occurred only in subjects accompanied by non-conditioned associates and observer associates. In the high intensity protocol, the social buffering effect occurred in subjects accompanied by either conditioned or non-conditioned associates, although the effect was more evident in the presence of non-conditioned subjects. Treatment of the conditioned associates with diazepam did not improve the social buffering effect. Moreover, social buffering effects were not correlated with self-grooming or prosocial behaviors, which indicates that the presence of another animal might decrease freezing by promotion of exploratory activity. Finally, the social buffering effect was not observed in the extinction test, either because the extinction was too effective in the moderate intensity protocol or because the extinction was equally ineffective in the high intensity protocol. Our results suggest that social buffering does not improve fear extinction consolidation.

From safety to frustration: The neural substrates of inhibitory learning in aversive and appetitive conditioning procedures.

Inhibitory associative learning counters the effects of excitatory learning, whether appetitively or aversively motivated. Moreover, the affective responses accompanying the inhibitory associations are of opponent valence to the excitatory conditioned responses. Inhibitors for negative aversive outcomes (e.g. shock) signal safety, while inhibitors for appetitive outcomes (e.g. food reward) elicit frustration and/or disappointment. This raises the question as to whether studies using appetitive and aversive conditioning procedures should demonstrate the same neural substrates for inhibitory learning. We review the neural substrates of appetitive and aversive inhibitory learning as measured in different procedural variants and in the context of the underpinning excitatory conditioning on which it depends. The mesocorticolimbic dopamine pathways, retrosplenial cortex and hippocampus are consistently implicated in inhibitory learning. Further neural substrates identified in some procedural variants may be related to the specific motivation of the learning task and modalities of the learning cues. Finally, we consider the translational implications of our understanding of the neural substrates of inhibitory learning, for obesity and addictions as well as for anxiety disorders.

Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory.

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

Interactions between human orbitofrontal cortex and hippocampus support model-based inference.

Internal representations of relationships between events in the external world can be utilized to infer outcomes when direct experience is lacking. This process is thought to involve the orbitofrontal cortex (OFC) and hippocampus (HPC), but there is little evidence regarding the relative role of these areas and their interactions in inference. Here, we used a sensory preconditioning task and pattern-based neuroimaging to study this question. We found that associations among value-neutral cues were acquired in both regions during preconditioning but that value-related information was only represented in the OFC at the time of the probe test. Importantly, inference was accompanied by representations of associated cues and inferred outcomes in the OFC, as well as by increased HPC-OFC connectivity. These findings suggest that the OFC and HPC represent only partially overlapping information and that interactions between the two regions support model-based inference.

SYNPLA, a method to identify synapses displaying plasticity after learning.

Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (synaptic proximity ligation assay), a synapse-specific, high-throughput, and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.

Representation of Fear of Heights by Basolateral Amygdala Neurons.

Fear of heights is evolutionarily important for survival, yet it is unclear how and which brain regions process such height threats. Given the importance of the basolateral amygdala (BLA) in mediating both learned and innate fear, we investigated how BLA neurons may respond to high-place exposure in freely behaving male mice. We found that a discrete set of BLA neurons exhibited robust firing increases when the mouse was either exploring or placed on a high place, accompanied by increased heart rate and freezing. Importantly, these high-place fear neurons were only activated under height threats, but not looming, acoustic startle, predatory odor, or mild anxiogenic conditions. Furthermore, after a fear-conditioning procedure, these high-place fear neurons developed conditioned responses to the context, but not the cue, indicating a convergence in processing of dangerous/risky contextual information. Our results provide insights into the neuronal representation of the fear of heights and may have implications for the treatment of excessive fear disorders.SIGNIFICANCE STATEMENT Fear can be innate or learned, as innate fear does not require any associative learning or experiences. Previous research mainly focused on studying the neural mechanism of learned fear, often using an associative conditioning procedure such as pairing a tone with a footshock. Only recently scientists started to investigate the neural circuits of innate fear, including the fear of predator odors and looming visual threats; however, how the brain processes the innate fear of heights is unclear. Here we provide direct evidence that the basolateral amygdala (BLA) is involved in representing the fear of heights. A subpopulation of BLA neurons exhibits a selective response to height and contextual threats, but not to other fear-related sensory or anxiogenic stimuli.

Human Sensory Cortex Contributes to the Long-Term Storage of Aversive Conditioning.

Growing animal data evince a critical role of the sensory cortex in the long-term storage of aversive conditioning, following acquisition and consolidation in the amygdala. Whether and how this function is conserved in the human sensory cortex is nonetheless unclear. We interrogated this question in a human aversive conditioning study using multidimensional assessments of conditioning and long-term (15 d) retention. Conditioned stimuli (CSs; Gabor patches) were calibrated to differentially activate the parvocellular (P) and magnocellular (M) visual pathways, further elucidating cortical versus subcortical mechanisms. Full-blown conditioning and long-term retention emerged for M-biased CS (vs limited effects for P-biased CS), especially among anxious individuals, in all four dimensions assessed: threat appraisal (threat ratings), physiological arousal (skin conductance response), perceptual learning [discrimination sensitivity (d') and response speed], and cortical plasticity [visual evoked potentials (VEPs) and cortical current density]. Interestingly, while behavioral, physiological, and VEP effects were comparable at immediate and delayed assessments, the cortical substrates evolved markedly over time, transferring from high-order cortices [inferotemporal/fusiform cortex and orbitofrontal cortex (OFC)] immediately to the primary and secondary visual cortex after the delay. In sum, the contrast between P- and M-biased conditioning confirms privileged conditioning acquisition via the subcortical pathway while the immediate cortical plasticity lends credence to the triadic amygdala-OFC-fusiform network thought to underlie threat processing. Importantly, long-term retention of conditioning in the basic sensory cortices supports the conserved role of the human sensory cortex in the long-term storage of aversive conditioning.SIGNIFICANCE STATEMENT A growing network of neural substrates has been identified in threat learning and memory. The sensory cortex plays a key role in long-term threat memory in animals, but such a function in humans remains unclear. To explore this problem, we conducted multidimensional assessments of immediate and delayed (15 d) effects of human aversive conditioning. Behavioral, physiological, and scalp electrophysiological data demonstrated conditioning effects and long-term retention. High-density EEG intracranial source analysis further revealed the cortical underpinnings, implicating high-order cortices immediately and primary and secondary visual cortices after the long delay. Therefore, while high-order cortices support aversive conditioning acquisition (i.e., threat learning), the human sensory cortex (akin to the animal homolog) underpins long-term storage of conditioning (i.e., long-term threat memory).

HDAC3 Activity within the Nucleus Accumbens Regulates Cocaine-Induced Plasticity and Behavior in a Cell-Type-Specific Manner.

Epigenetic mechanisms regulate processes of neuroplasticity critical to cocaine-induced behaviors. This includes the Class I histone deacetylase (HDAC) HDAC3, known to act as a negative regulator of cocaine-associated memory formation within the nucleus accumbens (NAc). Despite this, it remains unknown how cocaine alters HDAC3-dependent mechanisms. Here, we profiled HDAC3 expression and activity in total NAc mouse tissue following cocaine exposure. Although chronic cocaine did not affect expression of Hdac3 within the NAc, chronic cocaine did affect promoter-specific changes in HDAC3 and H4K8Ac occupancy. These changes in promoter occupancy correlated with cocaine-induced changes in expression of plasticity-related genes. To causally determine whether cocaine-induced plasticity is mediated by HDAC3's deacetylase activity, we overexpressed a deacetylase-dead HDAC3 point mutant (HDAC3-Y298H-v5) within the NAc of adult male mice. We found that disrupting HDAC3's enzymatic activity altered selective changes in gene expression and synaptic plasticity following cocaine exposure, despite having no effects on cocaine-induced behaviors. In further assessing HDAC3's role within the NAc, we observed that chronic cocaine increases Hdac3 expression in Drd1 but not Drd2-cells of the NAc. Moreover, we discovered that HDAC3 acts selectively within D1R cell-types to regulate cocaine-associated memory formation and cocaine-seeking. Overall, these results suggest that cocaine induces cell-type-specific changes in epigenetic mechanisms to promote plasticity important for driving cocaine-related behaviors.SIGNIFICANCE STATEMENT Drugs of abuse alter molecular mechanisms throughout the reward circuitry that can lead to persistent drug-associated behaviors. Epigenetic regulators are critical drivers of drug-induced changes in gene expression. Here, we demonstrate that the activity of an epigenetic enzyme promotes neuroplasticity within the nucleus accumbens (NAc) critical to cocaine action. In addition, we demonstrate that these changes in epigenetic activity drive cocaine-seeking behaviors in a cell-type-specific manner. These findings are key in understanding and targeting cocaine's impact of neural circuitry and behavior.

Basal forebrain projections to the lateral habenula modulate aggression reward.

Maladaptive aggressive behaviour is associated with a number of neuropsychiatric disorders and is thought to result partly from the inappropriate activation of brain reward systems in response to aggressive or violent social stimuli. Nuclei within the ventromedial hypothalamus, extended amygdala and limbic circuits are known to encode initiation of aggression; however, little is known about the neural mechanisms that directly modulate the motivational component of aggressive behaviour. Here we established a mouse model to measure the valence of aggressive inter-male social interaction with a smaller subordinate intruder as reinforcement for the development of conditioned place preference (CPP). Aggressors develop a CPP, whereas non-aggressors develop a conditioned place aversion to the intruder-paired context. Furthermore, we identify a functional GABAergic projection from the basal forebrain (BF) to the lateral habenula (lHb) that bi-directionally controls the valence of aggressive interactions. Circuit-specific silencing of GABAergic BF-lHb terminals of aggressors with halorhodopsin (NpHR3.0) increases lHb neuronal firing and abolishes CPP to the intruder-paired context. Activation of GABAergic BF-lHb terminals of non-aggressors with channelrhodopsin (ChR2) decreases lHb neuronal firing and promotes CPP to the intruder-paired context. Finally, we show that altering inhibitory transmission at BF-lHb terminals does not control the initiation of aggressive behaviour. These results demonstrate that the BF-lHb circuit has a critical role in regulating the valence of inter-male aggressive behaviour and provide novel mechanistic insight into the neural circuits modulating aggression reward processing.

Optogenetic reactivation of memory ensembles in the retrosplenial cortex induces systems consolidation.

The neural circuits underlying memory change over prolonged periods after learning, in a process known as systems consolidation. Postlearning spontaneous reactivation of memory-related neural ensembles is thought to mediate this process, although a causal link has not been established. Here we test this hypothesis in mice by using optogenetics to selectively reactivate neural ensembles representing a contextual fear memory (sometimes referred to as engram neurons). High-frequency stimulation of these ensembles in the retrosplenial cortex 1 day after learning produced a recent memory with features normally observed in consolidated remote memories, including higher engagement of neocortical areas during retrieval, contextual generalization, and decreased hippocampal dependence. Moreover, this effect was only present if memory ensembles were reactivated during sleep or light anesthesia. These results provide direct support for postlearning memory ensemble reactivation as a mechanism of systems consolidation, and show that this process can be accelerated by ensemble reactivation in an unconscious state.

The separate and combined effects of a dangerous context and an epinephrine injection on sensory preconditioning in rats.

Four experiments examined the effects of a dangerous context and a systemic epinephrine injection on sensory preconditioning in rats. In each experiment, rats were exposed to presentations of a tone and light in stage 1, light-shock pairings in stage 2, and test presentations of the tone alone and light alone in stage 3. Presentations of the tone and light in stage 1 occurred in either a safe or a previously shocked context, and/or under a systemic injection of epinephrine. Experiment 1 showed that a trace interval of 20 sec between presentations of the tone and light produced sensory preconditioning of the tone in a previously shocked context but not in a safe context, while experiment 2 provided evidence that this trace preconditioning was associative, due to the formation of a tone-light association. Experiment 3 showed that, in a safe context, exposure to the trace protocol under the influence of an epinephrine injection also produced sensory preconditioning of the tone, while experiment 4 provided evidence that a shocked context and an epinephrine injection have additive effects on trace preconditioning. These findings are discussed in relation to theories of trace conditioning. They suggest that the release of epinephrine by danger enhances attention and/or working memory processes, and thereby associative formation across a trace interval.

History of conditioned reward association disrupts inhibitory control: an examination of neural correlates.

The neural processes that support inhibitory control in the face of stimuli with a history of reward association are not yet well understood. Yet, the ability to flexibly adapt behavior to changing reward-contingency contexts is important for daily functioning and warrants further investigation. This study aimed to characterize neural and behavioral impacts of stimuli with a history of conditioned reward association on motor inhibitory control in healthy young adults by investigating group-level effects as well as individual variation in the ability to inhibit responses to stimuli with a reward history. Participants (N = 41) first completed a reward conditioning phase, during which responses to rewarded stimuli were associated with money and responses to unrewarded stimuli were not. Rewarded and unrewarded stimuli from training were carried forward as No-Go targets in a subsequent go/no-go task to test the effect of reward history on inhibitory control. Participants underwent functional brain imaging during the go/no-go portion of the task. On average, a history of reward conditioning disrupted inhibitory control. Compared to inhibition of responses to stimuli with no reward history, trials that required inhibition of responses to previously rewarded stimuli were associated with greater activity in frontal and striatal regions, including the inferior frontal gyrus, insula, striatum, and thalamus. Activity in the insula and thalamus during false alarms and in the ventromedial prefrontal cortex during correctly withheld trials predicted behavioral performance on the task. Overall, these results suggest that reward history serves to disrupt inhibitory control and provide evidence for diverging roles of the insula and ventromedial prefrontal cortex while inhibiting responses to stimuli with a reward history.

Fear extinction learning modulates large-scale brain connectivity.

Exploring the neural circuits of the extinction of conditioned fear is critical to advance our understanding of fear- and anxiety-related disorders. The field has focused on examining the role of various regions of the medial prefrontal cortex, insular cortex, hippocampus, and amygdala in conditioned fear and its extinction. The contribution of this 'fear network' to the conscious awareness of fear has recently been questioned. And as such, there is a need to examine higher/multiple cortical systems that might contribute to the conscious feeling of fear and anxiety. Herein, we studied functional connectivity patterns across the entire brain to examine the contribution of multiple networks to the acquisition of fear extinction learning and its retrieval. We conducted trial-by-trial analyses on data from 137 healthy participants who underwent a two-day fear conditioning and extinction paradigm in a functional magnetic resonance imaging (fMRI) scanner. We found that functional connectivity across a broad range of brain regions, many of which are part of the default mode, frontoparietal, and ventral attention networks, increased from early to late extinction learning only to a conditioned cue. The increased connectivity during extinction learning predicted the magnitude of extinction memory tested 24 h later. Together, these findings provide evidence supporting recent studies implicating distributed brain regions in learning, consolidation and expression of fear extinction memory in the human brain.

How expectations of pain elicited by consciously and unconsciously perceived cues unfold over time.

Expectation can shape the perception of pain within a fraction of time, but little is known about how perceived expectation unfolds over time and modulates pain perception. Here, we combine magnetoencephalography (MEG) and machine learning approaches to track the neural dynamics of expectations of pain in healthy participants with both sexes. We found that the expectation of pain, as conditioned by facial cues, can be decoded from MEG as early as 150 ms and up to 1100 ms after cue onset, but decoding expectation elicited by unconsciously perceived cues requires more time and decays faster compared to consciously perceived ones. Also, results from temporal generalization suggest that neural dynamics of decoding cue-based expectation were predominately sustained during cue presentation but transient after cue presentation. Finally, although decoding expectation elicited by consciously perceived cues were based on a series of time-restricted brain regions during cue presentation, decoding relied on the medial prefrontal cortex and anterior cingulate cortex after cue presentation for both consciously and unconsciously perceived cues. These findings reveal the conscious and unconscious processing of expectation during pain anticipation and may shed light on enhancing clinical care by demonstrating the impact of expectation cues.

Fear generalization immediately after contextual fear memory consolidation in mice.

Fear generalization is a symptom of anxiety-related disorders, including acute stress disorder and post-traumatic stress disorder. Using a contextual fear conditioning paradigm, we found that mice exposed to a similar neutral context but not a different neutral context soon after training showed fear generalization immediately after contextual fear memory consolidation (i.e., 6 h after training). This fear generalization was reflected by a change not only in the total amount but also the pattern of freezing between conditioned and generalized contexts. These results provide insight into the factors that influence fear generalization and can facilitate future studies investigating the underlying pathophysiological mechanisms of anxiety-related disorders.