Aversive Research Articles

Dissociable hindbrain GLP1R circuits for satiety and aversion.

The most successful obesity therapeutics, glucagon-like peptide-1 receptor (GLP1R) agonists, cause aversive responses such as nausea and vomiting1,2, effects that may contribute to their efficacy. Here, we investigated the brain circuits that link satiety to aversion, and unexpectedly discovered that the neural circuits mediating these effects are functionally separable. Systematic investigation across drug-accessible GLP1R populations revealed that only hindbrain neurons are required for the efficacy of GLP1-based obesity drugs. In vivo two-photon imaging of hindbrain GLP1R neurons demonstrated that most neurons are tuned to either nutritive or aversive stimuli, but not both. Furthermore, simultaneous imaging of hindbrain subregions indicated that area postrema (AP) GLP1R neurons are broadly responsive, whereas nucleus of the solitary tract (NTS) GLP1R neurons are biased towards nutritive stimuli. Strikingly, separate manipulation of these populations demonstrated that activation of NTSGLP1R neurons triggers satiety in the absence of aversion, whereas activation of APGLP1R neurons triggers strong aversion with food intake reduction. Anatomical and behavioural analyses revealed that NTSGLP1R and APGLP1R neurons send projections to different downstream brain regions to drive satiety and aversion, respectively. Importantly, GLP1R agonists reduce food intake even when the aversion pathway is inhibited. Overall, these findings highlight NTSGLP1R neurons as a population that could be selectively targeted to promote weight loss while avoiding the adverse side effects that limit treatment adherence.

Context-dependent hyperactivity in syngap1a and syngap1b zebrafish models of SYNGAP1-related disorder.

SYNGAP1-related disorder (SYNGAP1-RD) is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild-type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab mutant larvae are hyperactive compared to wild-type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, and hyperactivity was proportional to the number of mutant syngap1 alleles. Syngap1 loss-of-function mutations produce relatively subtle phenotypes in zebrafish compared to mammals. For example, while mouse Syngap1 homozygotes die at birth, zebrafish syngap1ab-/- survive to adulthood and are fertile, thus some aspects of symptoms in people with SYNGAP1-Related Disorder are not likely to be reflected in zebrafish. Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.

Distinct populations suppress or escalate intake of cocaine paired with aversive quinine.

Only a subset of individuals who encounter drugs of abuse become habitual users. Aversive subjective effects like coughing and unpleasant taste are predictors for continued use. While several preclinical studies have explored self-administration involving aversive cues, none have simultaneously introduced aversion with the initial drug self-administration. We aimed to develop a clinically relevant model by pairing intravenous cocaine with intraoral quinine self-administration from the outset and investigating whether repeated exposure to an aversive stimulus would alter its hedonic value under laboratory conditions. Twenty-seven male and female Sprague Dawley rats self-administered intravenous/intraoral (cocaine/quinine) for 2 hr/day over 14 days. This was followed by a 1-day quinine-only extinction session, a 3-day return to self-administration, and an intraoral infusion session to assess quinine taste reactivity (TR). We identified three distinct groups. The first self-administered very little cocaine, while the second sharply escalated cocaine intake. Both groups had similar aversive TR to quinine, suggesting that the escalating group did not habituate to the aversive cue but pursued drug despite it. We also identified a third group with high initial intake that decreased over time. This decrease predicted high aversive TR, and we argue this group may represent individuals who "overindulge" on their first use and subsequently find self-administration to be aversive. Our novel model mimics real-world variability in initial interactions with drugs of abuse and yields three distinct groups that differ in self-administration patterns and aversive cue valuation.

Defensive plasticity in South American rattlesnakes Crotalus durissus

Reptiles exhibit defensive responses in diverse ways, influenced by situational factors. Snakes, such as the South American rattlesnake Crotalus durissus, display complex defensive responses, adapting tactics based on circumstances when encountering potential predators. Environmental and intrinsic parameters shape snake behaviour during predator encounters. This study employs distinct stimuli to investigate behavioural changes and whether snakes adapt defensive strategies. We compare the behaviours of rattlesnakes before and after an aversive stimulus involving physical contact. Following encounters with higher-risk stimuli, snakes become more defensive.Findings reveal that snakes alter strategies based on threat intensity. This phenomenon might be influenced by the potential cost of defense and prior interactions with predators. The study underscores the intricate nature of snake defensive behaviour. Keywords: behavioural plasticity, antipredator behaviour, aversive stimulus, behavioural cost, active behaviours

Sensory over-responsivity and atypical neural responses to socially relevant stimuli in autism.

Although aversive responses to sensory stimuli are common in autism spectrum disorder (ASD), it remains unknown whether the social relevance of aversive sensory inputs affects their processing. We used functional magnetic resonance imaging (fMRI) to investigate neural responses to mildly aversive nonsocial and social sensory stimuli as well as how sensory over-responsivity (SOR) severity relates to these responses. Participants included 21 ASD and 25 typically-developing (TD) youth, aged 8.6-18.0 years. Results showed that TD youth exhibited significant neural discrimination of socially relevant versus irrelevant aversive sensory stimuli, particularly in the amygdala and orbitofrontal cortex (OFC), regions that are crucial for sensory and social processing. In contrast, ASD youth showed reduced neural discrimination of social versus nonsocial stimuli in the amygdala and OFC, as well as overall greater neural responses to nonsocial compared with social stimuli. Moreover, higher SOR in ASD was associated with heightened responses in sensory-motor regions to socially-relevant stimuli. These findings further our understanding of the relationship between sensory and social processing in ASD, suggesting limited attention to the social relevance compared with aversiveness level of sensory input in ASD versus TD youth, particularly in ASD youth with higher SOR.

Distinct Mindfulness States Produce Dissociable Effects on Neural Markers of Emotion Processing: Evidence From the Late Positive Potential

BackgroundMindfulness has long been theorized to benefit emotion regulation, but despite the ubiquity of the claim, there is little empirical evidence demonstrating how mindfulness modulates the neurophysiology of emotion processing. The current study aimed to fill this gap in knowledge by leveraging a novel research approach capable of discretizing mindfulness into distinct states of open monitoring (OM) and focused attention (FA) to distinguish their influence on multimodal subjective and objective measures of emotion processing. MethodsUtilizing a fully within-participant picture viewing state induction protocol (N = 30), we compared the effects of OM and FA, rigorously contrasted against an active control, on the visually evoked late positive potential (LPP), a neural index of motivated attention. Bayesian mixed modeling was used to distinguish OM versus FA effects on the early and late sustained LPP while evaluating the influence of subjective arousal ratings as a within-participant moderator of the state inductions. ResultsWhen negative picture trials were retrospectively rated as more subjectively arousing, the OM induction reduced the late sustained LPP response, whereas the FA induction enhanced the LPP. ConclusionsAcute manipulation of OM and FA states may reduce and enhance motivated attention to aversive stimuli during conditions of high subjective arousal, respectively. Functional distinctions between different mindfulness states on emotion processing may be most dissociable after accounting for within-participant variability in how stimuli are appraised. These results support the future potential of the state induction protocol for parsing the neural affective mechanisms that underlie mindfulness training programs and interventions.

A REM-active basal ganglia circuit that regulates anxiety

Rapid eye movement (REM) sleep has been hypothesized to promote emotional resilience, but any neuronal circuits mediating this have not been identified. We find that in mice, somatostatin (Som) neurons in the entopeduncular nucleus (EPSom)/internal globus pallidus are predominantly active during REM sleep. This unique REM activity is both necessary and sufficient for maintaining normal REM sleep. Inhibiting or exciting EPSom neurons reduced or increased REM sleep duration, respectively. Activation of the sole downstream target of EPSom neurons, Vglut2 cells in the lateral habenula (LHb), increased sleep via the ventral tegmental area (VTA). A simple chemogenetic scheme to periodically inhibit the LHb over 4days selectively removed a significant amount of cumulative REM sleep. Chronic, but not acute, REM reduction correlated with mice becoming anxious and more sensitive to aversive stimuli. Therefore, we suggest that cumulative REM sleep, in part generated by the EP → LHb → VTA circuit identified here, could contribute to stabilizing reactions to habitual aversive stimuli.

From mistakes to mastery: An idiographic microanalytical study comparing processes related to effective and ineffective outcomes in behavioral interventions

The field of psychotherapy research increasingly recognizes the need for idiographic studies linking process of change to outcomes. However, investigations have typically focused on successful outcomes alone, neglecting the analysis of specific behaviors connected to treatment failure. This study aims to link process to both effective and ineffective outcomes. By examining 80 psychotherapy sessions across 13 cases with a mixed-method approach, a turn-by-turn conversational analysis was carried out. After detecting different levels of effectiveness following a multiple baseline design, sequential analysis and intra- and inter-case comparisons were performed. The results indicated that while differential reinforcement of target behavior was found to be related to both successful and unsuccessful outcomes, the defining characteristics of effective sessions were the preciseness of specific strategies and the predominance of appetitive over aversive stimulation throughout the intervention. These findings revealed that splitting units into linear relations to analyze psychotherapeutic interaction might be insufficient. Instead, addressing not only the presence but also the absence and concurrence of covariation associated with the events of interest could be a determining factor in unveiling the processes leading to different outcomes in psychotherapy.

Increased reactivity of the paraventricular nucleus of the hypothalamus and decreased threat responding in male rats following psilocin administration

Psychedelics have experienced renewed interest following positive clinical effects, however the neurobiological mechanisms underlying effects remain unclear. The paraventricular nucleus of the hypothalamus (PVN) plays an integral role in stress response, autonomic function, social behavior, and other affective processes. We investigated the effect of psilocin, the psychoactive metabolite of psilocybin, on PVN reactivity in Sprague Dawley rats. Psilocin increased stimulus-independent PVN activity as measured by c-Fos expression in male and female rats. Psilocin increased PVN reactivity to an aversive air-puff stimulus in males but not females. Reactivity was restored at 2- and 7-days post-injection with no group differences. Additionally, prior psilocin injection did not affect PVN reactivity following acute restraint stress. Experimental groups sub-classified by baseline threat responding indicate that increased male PVN reactivity is driven by active threat responders. These findings identify the PVN as a significant site of psychedelic drug action with implications for threat responding behavior.

Predatory Odor Exposure as a Potential Paradigm for Studying Emotional Modulation of Memory Consolidation-The Role of the Noradrenergic Transmission in the Basolateral Amygdala.

The pivotal role of the basolateral amygdala (BLA) in the emotional modulation of hippocampal plasticity and memory consolidation is well-established. Specifically, multiple studies have demonstrated that the activation of the noradrenergic (NA) system within the BLA governs these modulatory effects. However, most current evidence has been obtained by direct infusion of synthetic NA or beta-adrenergic agonists. In the present study, we aimed to investigate the effect of endogenous NA release in the BLA, induced by a natural aversive stimulus (coyote urine), on memory consolidation for a low-arousing, hippocampal-dependent task. Our experiments combined a weak object location task (OLT) version with subsequent mild predator odor exposure (POE). To investigate the role of endogenous NA in the BLA in memory modulation, a subset of the animals (Wistar rats) was treated with the non-selective beta-blocker propranolol at the end of the behavioral procedures. Hippocampal tissue was collected 90 min after drug infusion or after the OLT test, which was performed 24 h later. We used the obtained samples to estimate the levels of phosphorylated CREB (pCREB) and activity-regulated cytoskeleton-associated protein (Arc)-two molecular markers of experience-dependent changes in neuronal activity. The result suggests that POE has the potential to become a valuable behavioral paradigm for studying the interaction between BLA and the hippocampus in memory prioritization and selectivity.

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Burst firing in Output-Defined Parallel Habenula Circuit Underlies the Antidepressant Effects of Bright Light Treatment.

Research highlights the significance of increased bursting in lateral habenula (LHb) neurons in depression and as a focal point for bright light treatment (BLT). However, the precise spike patterns of LHb neurons projecting to different brain regions during depression, their roles in depression development, and BLT's therapeutic action remain elusive. Here, LHb neurons are found projecting to the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and median raphe nucleus (MnR) exhibit increased bursting following aversive stimuli exposure, correlating with distinct depressive symptoms. Enhanced bursting in DRN-projecting LHb neurons is pivotal for anhedonia and anxiety, while concurrent bursting in LHb neurons projecting to the DRN, VTA, and MnR is essential for despair. Remarkably, reducing bursting in distinct LHb neuron subpopulations underlies the therapeutic effects of BLT on specific depressive behaviors. These findings provide valuable insights into the mechanisms of depression and the antidepressant action of BLT.

Parvalbumin-expressing basal forebrain neurons mediate learning from negative experience

Parvalbumin (PV)-expressing GABAergic neurons of the basal forebrain (BFPVNs) were proposed to serve as a rapid and transient arousal system, yet their exact role in awake behaviors remains unclear. We performed bulk calcium measurements and electrophysiology with optogenetic tagging from the horizontal limb of the diagonal band of Broca (HDB) while male mice were performing an associative learning task. BFPVNs responded with a distinctive, phasic activation to punishment, but showed slower and delayed responses to reward and outcome-predicting stimuli. Optogenetic inhibition during punishment impaired the formation of cue-outcome associations, suggesting a causal role of BFPVNs in associative learning. BFPVNs received strong inputs from the hypothalamus, the septal complex and the median raphe region, while they synapsed on diverse cell types in key limbic structures, where they broadcasted information about aversive stimuli. We propose that the arousing effect of BFPVNs is recruited by aversive stimuli to serve crucial associative learning functions.

Medial prefrontal cortex suppresses reward-seeking behavior with risk of punishment by reducing sensitivity to reward.

Reward-seeking behavior is frequently associated with risk of punishment. There are two types of punishment: positive punishment, which is defined as addition of an aversive stimulus, and negative punishment, involves the omission of a rewarding outcome. Although the medial prefrontal cortex (mPFC) is important in avoiding punishment, whether it is important for avoiding both positive and negative punishment and how it contributes to such avoidance are not clear. In this study, we trained male mice to perform decision-making tasks under the risks of positive (air-puff stimulus) and negative (reward omission) punishment, and modeled their behavior with reinforcement learning. Following the training, we pharmacologically inhibited the mPFC. We found that pharmacological inactivation of mPFC enhanced the reward-seeking choice under the risk of positive, but not negative, punishment. In reinforcement learning models, this behavioral change was well-explained as an increase in sensitivity to reward, rather than a decrease in the strength of aversion to punishment. Our results suggest that mPFC suppresses reward-seeking behavior by reducing sensitivity to reward under the risk of positive punishment.

Evaluation of ivermectin and vitamin E based combination with antiseizure rufinamide drug for mitigation of pentylenetetrazole-induced kindling, behavioral challenges and histopathological aberrations.

Pentylenetetrazole- (PTZ)-induced kindling is a broadly used experimental model to evaluate the impact of antiseizure drugs and their novel combination on seizure progression. The current study aimed to evaluate the anti-kindling effects of ivermectin (IVM) and rufinamide (RUFI) alone and their combination with vitamin E. The mice were administered 11 injections of PTZ (40 mg/kg) followed by assessment for anxiety-like behavior and cognitive abilities in a series of behavior tests with subsequent brain isolation for biochemical and histopathological evaluation. The outcomes showed a marked protection by IVM + RUFI (P<0.001) from kindling progression, anxiety-like behavior and cognitive deficit. However, additional supplementation with vitamin E worked superior to duo therapy as these mice were noted to be most fearless to visiting open, illuminated and elevated zones of open field, light/dark and elevated-plus maze (P<0.0001). Further, they showed marked remembrance of the familiar milieu in y-maze (P<0.01) and novel objection recognition (P<0.05) tests. Additionally, their recollection of aversive stimuli in passive avoidance and spatial memory in Morris water maze were evident (P<0.0001), in comparison to kindled mice. The IVM + RUFI duo therapy and its co-administration with vitamin E prevented kindling-triggered oxidative stress in brains and neuronal damage in hippocampus. We conclude that the benefits of the co-administration of vitamin E might be the results of antioxidant and anti-inflammatory effects of vitamin E which might be potentiating the antiseizure effects of RUFI and GABA-A modulating potential by ivermectin.

The effect of late-life environmental enrichment on stress and anxiety: The role of sex and age-related differences in coping with aversive stimuli

Anxiety significantly diminishes the quality of life in older adults, and the drugs used for its treatment often come with risky side effects. Non-pharmacological protocols could be valuable, but more research is needed in this area. Environmental enrichment induces positive effects on anxiety-like behavior in young and adult animals; whether the same happens in aged animals is still elusive. The aged brain undergoes changes that contribute to make it “fragile” and consequently even mild, potentially positive stimuli can trigger dyshomeostasis, worsening rather than ameliorating functioning. Here, by combining behavioral analysis and measurement of serum and brain corticosterone levels, we show that late-life environmental enrichment can induce eustress or distress, depending on sex and hypothalamic-pituitary-adrenal axis function. These findings pave the way for optimizing outcomes and minimizing undesired effects in the clinical setting, underscoring the need to overcome the limits of gender medicine and emphasizing the crucial role of individually tailored therapies.

A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Surprising multifunctionality of a Tritonia swim CPG neuron: C2 drives the early phase of postswim crawling despite being silent during the behavior.

In response to a suitably aversive skin stimulus, the marine mollusk Tritonia diomedea launches an escape swim followed by several minutes of high-speed crawling. The two escape behaviors are highly dissimilar: whereas the swim is a muscular behavior involving alternating ventral and dorsal whole body flexions, the crawl is a nonrhythmic gliding behavior mediated by the beating of foot cilia. The serotonergic dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and also strongly drive crawling. Although the swim network is very well understood, the Tritonia crawling network to date comprises only three neurons: the DSIs and pedal neurons 5 and 21 (Pd5 and Pd21). Since Tritonia's swim network has been suggested to have arisen from a preexisting crawling network, we examined the possible role that another swim CPG neuron, C2, may play in crawling. Because of its complete silence in the postswim crawling period, C2 had not previously been considered to play a role in driving crawling. However, semi-intact preparation experiments demonstrated that a brief C2 spike train surprisingly and strongly drives the foot cilia for ∼30 s, something that cannot be explained by its synaptic connections to Pd5 and Pd21. Voltage-sensitive dye (VSD) imaging in the pedal ganglion identified many candidate crawling motor neurons that fire at an elevated rate after the swim and also revealed several pedal neurons that are strongly excited by C2. It is intriguing that unlike the DSIs, which fire tonically after the swim to drive crawling, C2 does so despite its postswim silence.NEW & NOTEWORTHY Tritonia swim central pattern generator (CPG) neuron C2 surprisingly and strongly drives the early phase of postswim crawling despite being silent during this period. In decades of research, C2 had not been suspected of driving crawling because of its complete silence after the swim. Voltage-sensitive dye imaging revealed that the Tritonia crawling motor network may be much larger than previously known and also revealed that many candidate crawling neurons are excited by C2.

Ventral pallidum GABA and glutamate neurons drive approach and avoidance through distinct modulation of VTA cell types.

The ventral pallidum (VP) contains GABA and glutamate neurons projecting to ventral tegmental area (VTA) whose stimulation drives approach and avoidance, respectively. Yet little is known about the mechanisms by which VP cell types shape VTA activity and drive behavior. Here, we found that both VP GABA and glutamate neurons were activated during approach to reward or by delivery of an aversive stimulus. Stimulation of VP GABA neurons inhibited VTA GABA, but activated dopamine and glutamate neurons. Remarkably, stimulation-evoked activation was behavior-contingent such that VTA recruitment was inhibited when evoked by the subject's own action. Conversely, VP glutamate neurons activated VTA GABA, as well as dopamine and glutamate neurons, despite driving aversion. However, VP glutamate neurons evoked dopamine in aversion-associated ventromedial nucleus accumbens (NAc), but reduced dopamine releasein reward-associated dorsomedial NAc. These findings show how heterogeneous VP projections to VTA can be engaged to shape approach and avoidance behaviors.

Functional diversity of dopamine axons in prefrontal cortex during classical conditioning

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.