Conditioned Aversion Research Articles

Aversive conditioning, anxiety, and the strategic control of attention

ABSTRACT What we pay attention to is influenced by both reward learning and aversive conditioning. Although early attention tends to be biased toward aversively conditioned stimuli, sustained ignoring of such stimuli is also possible. How aversive conditioning influences how a person chooses to search, or the strategic control of attention, has not been explored. In the present study, participants learned an association between a colour and an aversive outcome during a training phase, and in a subsequent test phase searched for one of two targets presented on each trial; one target was rendered in the aversively conditioned colour (CS+) and the other in a neutral colour (CS-). Given the distribution of colour stimuli in the search array, it was more optimal to search for and report a target in one of the two colours on some trials. Our results demonstrate that participants were biased away from the CS+ target, which resulted in non-optimal search on some trials. Surprisingly, rather than accentuate this bias, greater state anxiety was associated with a stronger tendency to find and report the CS+ target. Our findings have implications for our understanding of the learning-dependent control of attention and abnormal attentional biases observed in high-anxious individuals.

Valenced tactile information is evoked by neutral visual cues following emotional learning

Abstract Learning which stimuli in our environment co-occur with painful or pleasurable events is critical for survival. Previous research has established the basic neural and behavioral mechanisms of aversive and appetitive conditioning; however, it is unclear precisely what information content is learned. Here we examined the degree to which aspects of the unconditioned stimulus (US)—sensory information versus affective salience—are transferred to the conditioned stimulus (CS). To decode what stimuli features (e.g., valence vs. discriminative somatosensation) are represented in patterns of brain activation elicited during appetitive (soft touch) and aversive (painful touch) conditioning to faces, a novel approach to using modeling with representational similarity analysis (RSA) based on theoretically driven representational patterns of interest (POIs) was applied to fMRI data. Once associations were learned through conditioning, globally, the CS reactivated US representational patterns showing conditioning-dependent reactivation in specific high-order brain regions: In the dorsal anterior cingulate cortex, the CS reactivated patterns associated with the affective salience of the US—suggesting that, with affective conditioning, these regions carry forward the affective associations of the experience.

Cortical kappa opioid receptors integrate negative affect and sleep disturbance

Sleep disruption and negative affect are attendant features of many psychiatric and neurological conditions that are often co-morbid including major depressive disorder, generalized anxiety disorder and chronic pain. Whether there is a causal relationship between negative affect and sleep disruption remains unclear. We therefore asked if mechanisms promoting negative affect can disrupt sleep and whether inhibition of pathological negative affect can normalize disrupted sleep. Signaling at the kappa opioid receptor (KOR) elicits dysphoria in humans and aversive conditioning in animals. We tested the possibility that (a) increased KOR signaling in the anterior cingulate cortex (ACC), a brain region associated with negative emotions, would be sufficient to promote both aversiveness and sleep disruption and (b) inhibition of KOR signaling would normalize pathological negative affect and sleep disruption induced by chronic pain. Chemogenetic Gi-mediated inhibition of KOR-expressing ACC neurons produced conditioned place aversion (CPA) as well as sleep fragmentation in naïve mice. CRISPR/Cas9 editing of ACC KOR normalized both the negative affect and sleep disruption elicited by pathological chronic pain while maintaining the physiologically critical sensory features of pain. These findings suggest therapeutic utility of KOR antagonists for treatment of disease conditions that are associated with both negative affect and sleep disturbances.

Ketamine modulates the exploratory dynamics and homebase-related behaviors of adult zebrafish

Anxiety is a protective behavior when animals face aversive conditions, but commonly associated with various neuropsychiatric disorders when exacerbated. Drug repurposing has emerged as a valuable strategy to use existing pharmaceuticals for new therapeutic purposes. Ketamine, traditionally used as an anesthetic, acts as a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, and evidence has shown potential anxiolytic and antidepressant effects at subanesthetic doses. However, the influence of ketamine on multiple behavioral domains in vertebrates is not completely understood. Here, we evaluated the potential modulatory effect of ketamine on the spatio-temporal exploratory dynamics and homebase-related behaviors in adult zebrafish using the open field test (OFT). Animals were exposed to subanesthetic concentrations of ketamine (0, 2, 20, and 40 mg/L) for 20 min and locomotion- exploration- and homebase-related behaviors were assessed in a single 30-min trial. Our data revealed that ketamine (20 and 40 mg/L) induced hyperlocomotion, as verified by the increased total distance traveled. All concentrations tested elicited circling behavior, a stereotyped-like response which gradually reduced across the periods of test. We also observed modulatory effects of ketamine on the spatio-temporal exploratory pattern, in which the reduced thigmotaxis and homebase activity, associated with the increased average length of trips were suggestive of anxiolytic-like effects. Collectively, our novel findings support the modulatory effects of ketamine on the spatio-temporal exploratory activity, as well as the utility of homebase-related measurements to evaluate the behavioral dynamics of zebrafish.

Contextual modulation of human associative learning following novelty-facilitated extinction, counterconditioning, and conventional extinction.

The expression of an association between a conditioned stimulus (CS) and an unconditioned stimulus (US) can be attenuated by presenting the CS by itself (i.e., extinction, Ext). Though effective, Ext is susceptible to recovery effects such as renewal, spontaneous recovery, and reinstatement. Dunsmoor et al. (2015, 2019) have proposed that pairing the CS with a neutral outcome (novelty-facilitated Ext [NFE]) could offer better protection against recovery effects than Ext. Though NFE has been compared to Ext, it has rarely been compared to counterconditioning (CC), a similar procedure except that the CS is paired with a US having a valence opposite to the US used in initial training. We report two aversive conditioning experiments using the rapid-trial streaming procedure with human participants that compare the efficacies and susceptibilities to ABA renewal of Ext, CC, and NFE. Associative learning was assessed through expectancy learning and evaluative conditioning. CC and NFE equally decreased anticipation of the US in the presence of the CS (i.e., expectancy learning). Depending on how the CS-US association was probed, they were either as or more effective at doing so than Ext. All three interference treatments were equally susceptible to context manipulations. Only CC clearly altered the valence of the CS (i.e., evaluative conditioning). Valence ratings after Ext, CC, and NFE, as well as a no-interference control condition, were all equally susceptible to context effects. Overall, the present study does not support the assertion that NFE is consistently more resistant to recovery effects than Ext. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Independent operations of appetitive and aversive conditioning systems lead to simultaneous production of conflicting memories in an insect.

Pavlovian conditioning is a ubiquitous form of associative learning that enables animals to remember appetitive and aversive experiences. Animals possess appetitive and aversive conditioning systems that memorize and retrieve appetitive and aversive experiences. Here, we addressed a question of whether integration of competing appetitive and aversive information takes place during the encoding of the experience or during memory retrieval. We developed novel experimental procedures to address this question using crickets (Gryllus bimaculatus), which allowed selective blockade of the expression of appetitive and aversive memories by injecting octopamine and dopamine receptor antagonists. We conditioned an odour (conditioned stimulus 1, CS1) with water and then with sodium chloride solution. At 24 h after conditioning, crickets retained both appetitive and aversive memories, and the memories were integrated to produce a conditioned response (CR). Importantly, when a visual pattern (CS2) was conditioned with CS1, appetitive and aversive memories formed simultaneously. This indicates that appetitive and aversive second-order conditionings are achieved at the same time. The memories were integrated for producing a conditioned response. We conclude that appetitive and aversive conditioning systems operate independently to form parallel appetitive and aversive memories, which compete to produce learned behaviour in crickets.

Valence and Salience Encoding in the Central Amygdala.

The central amygdala (CeA) has emerged as an important brain region for regulating both negative (fear and anxiety) and positive (reward) affective behaviors. The CeA has been proposed to encode affective information in the form of valence (whether the stimulus is good or bad) or salience (how significant is the stimulus), but the extent to which these two types of stimulus representation occur in the CeA is not known. Here, we used single cell calcium imaging in mice during appetitive and aversive conditioning and found that majority of CeA neurons (~65%) encode the valence of the unconditioned stimulus (US) with a smaller subset of cells (~15%) encoding the salience of the US. Valence and salience encoding of the conditioned stimulus (CS) was also observed, albeit to a lesser extent. These findings show that the CeA is a site of convergence for encoding oppositely valenced US information.

Prior Running, as Well as Swimming, Hinders Swimming‐Based Taste Aversion Learning in Rats

AbstractRunning in an exercise wheel or swimming in a water pool endows rats with a conditioned aversion to a taste substance consumed before the activity. The present study explores the psychophysiological processes underlying these activity‐based taste aversions. Experiment 1 demonstrated that prior experience of running or swimming (i.e., familiarization) hinders subsequent taste aversion learning based on swimming in rats, implying a similar psychophysiological process between running and swimming in establishing taste aversion. Experiment 2 replicated the findings of Experiment 1 with rats obtained from another animal supplier. The running‐to‐swimming cross‐familiarization effect demonstrated in these experiments contrasts with a previous report in which prior swimming had no effect on subsequent running‐based taste aversion learning. Theoretical considerations regarding such asymmetrical cross‐familiarization effects extended to discussions on the similarities and differences in the psychophysiological states induced by running, swimming, and emetic lithium chloride injection.

Histamine H3 receptor activation in the insular cortex during taste memory conditioning decreases appetitive response but accelerates aversive memory extinction under an ad libitum liquid regimen

Conditioned taste aversion (CTA) is a robust associative learning; liquid deprivation during this conditioning allows researchers to obtain readable measures of associative learning. Recent research suggests that thirst could be a crucial motivator that modulates conditioning and memory extinction processes, highlighting the importance of the body’s internal state during learning. Furthermore, the histaminergic system is one of the major modulatory systems controlling several behavioral and neurobiological functions, such as feeding, water intake, and nociception. Therefore, this research aimed to assess the effect of H3 histaminergic receptor activation in the insular cortex (IC) during CTA. For this, we conditioned adult male Wistar rats under two regimens: water deprivation and water ad libitum. A classical CTA protocol was used for water deprivation. Before CTA acquisition, 10 μM R-α-methylhistamine (RAMH), an H3 receptor agonist, was injected into the IC. Results showed that RAMH injections decreased CTA in water-deprived rats without affecting the significant aversion conditioning in rats that were given water ad libitum. Moreover, RAMH accelerated the process of aversive memory extinction under ad libitum water conditions. According to our findings, the degree of liquid satiety differentially affected taste-aversive memory formation, and H3 histamine receptors were more involved under water deprivation conditions during acquisition. However, these receptors modulated the strength of aversive conditioning by altering the rate of aversive memory extinction in the absence of deprivation. In conclusion, histaminergic activity in the IC may influence taste memory dynamics through different mechanisms depending on the degree of liquid satiety or deprivation during conditioning.

Retrosplenial cortex and aversive conditioning.

The retrosplenial cortex (RSC) is well-known for its contribution to episodic memory, as well as contextual and spatial learning and memory. However, two literatures have also emerged examining the role of the RSC in aversive conditioning. The purpose of this manuscript is to review, and attempt to integrate, these two literatures. We focus on studies in which discrete cues, such as tones, predict the occurrence of aversive outcomes, such as mild shocks. Using both electrophysiological recordings and lesion methods, the first literature has examined RSC contributions to discriminative avoidance conditioning. The second, and more recent literature, has focused on the role of the RSC in Pavlovian fear conditioning. We discuss both literatures in terms of the type of information processed by the RSC, the role of the RSC in memory storage, and how the aversive conditioning literature might be consistent with a role for the RSC in contextual learning and memory.

Mu-opioid receptor knockout on Foxp2-expressing neurons reduces aversion-resistant alcohol drinking.

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic marker of intercalated (ITC) cells in the amygdala and a subset of striatal medium spiny neurons (MSNs), both of which express MORs in wild-type mice and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. For the current series of studies, we characterized the behavior of mice with genetic deletion of the MOR gene Oprm1 in Foxp2-expressing neurons (Foxp2-Cre/Oprm1fl/fl). Male and female Foxp2-Cre/Oprm1fl/fl mice were generated and heterozygous Cre+ (knockout) and homozygous Cre- (control) animals were tested for aversion-resistant alcohol consumption using an intermittent access (IA) task, operant responding for a sucrose reward, conditioned place aversion (CPA) to morphine withdrawal, and locomotor sensitization to morphine. The results demonstrate that deletion of MOR on Foxp2-expressing neurons renders mice more sensitive to quinine-adulterated ethanol (EtOH). Mice with the deletion (vs. Cre- controls) also consumed less alcohol during the final sessions of the IA task, responded less for sucrose under an FR3 schedule, and were less active at baseline and following morphine injection. Foxp2-MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2-expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking.

Neurophysiological and Autonomic Dynamics of Threat Processing During Sustained Social Fear Generalization.

Survival in dynamic environments requires that organisms learn to predict danger from situational cues. One key facet of threat prediction is generalization from a predictive cue to similar cues, ensuring that a cue-outcome contingency is applied beyond the original learning environment. Generalization has been observed in laboratory studies of aversive conditioning: behavioral and physiological processes generalize responses from a stimulus paired with threat (the CS+) to unpaired stimuli, with response magnitudes varying with CS+ similarity. In contrast, work focusing on sensory responses in visual cortex has found a sharpening pattern, in which responses to stimuli closely resembling the CS+ are maximally suppressed, potentially reflecting lateral inhibitory interactions with the CS+ representation. Originally demonstrated with simple visual cues, changes in visuocortical tuning have also been observed in threat generalization learning across facial identities. It is unclear to what extent these visuocortical changes represent transient or sustained effects and if generalization learning requires prior conditioning to the CS+. The present study addressed these questions using EEG and pupillometry in an aversive generalization paradigm involving hundreds of trials using a gradient of facial identities. Visuocortical ssVEP sharpening occurred after dozens of trials of generalization learning without prior differential conditioning, but diminished as learning continued. By contrast, generalization of alpha power suppression, pupil dilation, and self-reported valence and arousal was seen throughout the experiment. Findings are consistent with threat processing models emphasizing the role of changing visucocortical and attentional dynamics when forming, curating, and shaping fear memories as observers continue learning about stimulus-outcome contingencies.

No impairment of contextual fear memory consolidation by oxytocin receptor antagonism in male rats

Oxytocin is a peptide released into brain regions associated with the processing of aversive memory and threat responses. Given the expression of oxytocin receptors across this vigilance surveillance system of the brain, we investigated whether pharmacological antagonism of the receptor would impact contextual aversive conditioning and memory. Adult male rats were conditioned to form an aversive contextual memory. The effects of peripheral administration of either the competitive antagonist Atosiban or noncompetitive antagonist L-368,899 were compared to saline controls. Oxytocin receptor antagonism treatment did not significantly impact the consolidation of aversive contextual memory in any of the groups. We conclude that peripheral antagonism of oxytocin signalling did not impact the formation of aversive memory.

Effects of morphine on conditioned place preference and pain are independent of uptake‑2.

Morphine changes neurotransmitter release, including norepinephrine, dopamine, and serotonin. Decynium‑22 (D22) inhibits an alternative neurotransmitter removal pathway, namely uptake‑2. Uptake‑2 includes plasma membrane monoamine transporter (PMAT) and organic cation transporters that have a low affinity, but high capacity for uptake of various monoamines such as norepinephrine, dopamine, and serotonin. This study was done to assess the effect of uptake‑2 inhibition on morphine‑induced conditioned place preference (CPP) and analgesia. In this study, the effects of morphine and/or D22 on CPP were evaluated following intraperitoneal injection in mice. Afterward, changes in motor activity were evaluated by the open field test. Using the tail‑flick model, the effects of D22 and/or morphine were evaluated on the pain threshold. The results showed that 20 mg/kg of morphine induced a place preference response. D22, at the dose of 0.03 mg/kg, caused place avoidance, while at the dose of 0.3 mg/kg, it produced a notable place preference response. Co‑administration of D22 and morphine showed that morphine reversed the CPP aversion induced by D22 at the lowest dose. Motor activity did not alter. In the tail‑flick test, morphine, at the dose of 3 mg/kg but not 1 mg/kg, increased the pain threshold. D22 induced significant analgesic responses. Co‑administration of D22 and morphine caused considerable analgesic effects. The findings revealed that D22 induced both conditioned aversion and preference depending on the dose while morphine induced CPP. Both drugs produced analgesia.

Preexposure to running attenuates rats' running-based flavour avoidance: Testing associative blocking with a cover cues or context change.

Voluntary running in activity wheels by rats leads to a Pavlovian conditioned aversion to the flavour consumed immediately before the running, causing the rats to avoid that flavour. This learning process, known as running-based flavour avoidance learning (FAL), is weakened when the rats have had repeated exposure to the wheels before. According to the associative account, the association between the background context and running established during the preexposure phase blocks the conditioning of the target flavour because the running is highly predictable by the background context from the outset of the FAL phase. Experiments 1 and 2 examined this account by introducing another flavour as a cue signalling wheel access during the preexposure phase. In the framework of the associative account, the introduction of this cue should impede the formation of the context-running association during the preexposure phase, thereby hindering the contextual blocking of aversive conditioning for the target flavour in the FAL phase. This would result in unweakened FAL. Although the results of Experiment 1 align with this prediction, in Experiment 2, when highly distinct flavours were used as the target and second cues, the preexposure effect was not eliminated. This contradicts the predictions of the associative account, indicating that Experiment 1 may have been influenced by stimulus generalisation. In Experiment 3, changing background contexts between the preexposure and FAL phases had no impact on the preexposure effect, contrary to the predictions of the associative account. In general, the associative account was not supported.

Auditory aversive generalization learning prompts threat-specific changes in alpha-band activity.

Pairing a neutral stimulus with aversive outcomes prompts neurophysiological and autonomic changes in response to the conditioned stimulus (CS+), compared to cues that signal safety (CS-). One of these changes-selective amplitude reduction of parietal alpha-band oscillations-has been reliably linked to processing of visual CS+. It is, however, unclear to what extent auditory conditioned cues prompt similar changes, how these changes evolve as learning progresses, and how alpha reduction in the auditory domain generalizes to similar stimuli. To address these questions, 55 participants listened to three sine wave tones, with either the highest or lowest pitch (CS+) being associated with a noxious white noise burst. A threat-specific (CS+) reduction in occipital-parietal alpha-band power was observed similar to changes expected for visual stimuli. No evidence for aversive generalization to the tone most similar to the CS+ was observed in terms of alpha-band power changes, aversiveness ratings, or pupil dilation. By-trial analyses found that selective alpha-band changes continued to increase as aversive conditioning continued, beyond when participants reported awareness of the contingencies. The results support a theoretical model in which selective alpha power represents a cross-modal index of continuous aversive learning, accompanied by sustained sensory discrimination of conditioned threat from safety cues.

Nucleus accumbens neurons dynamically respond to appetitive and aversive associative learning.

To survive, individuals must learn to associate cues in the environment with emotionally relevant outcomes. This association is partially mediated by the nucleus accumbens (NAc), a key brain region of the reward circuit that is mainly composed by GABAergic medium spiny neurons (MSNs), that express either dopamine receptor D1 or D2. Recent studies showed that both populations can drive reward and aversion, however, the activity of these neurons during appetitive and aversive Pavlovian conditioning remains to be determined. Here, we investigated the relevance of D1- and D2-neurons in associative learning, by measuring calcium transients with fiber photometry during appetitive and aversive Pavlovian tasks in mice. Sucrose was used as a positive valence unconditioned stimulus (US) and foot shock was used as a negative valence US. We show that during appetitive Pavlovian conditioning, D1- and D2-neurons exhibit a general increase in activity in response to the conditioned stimuli (CS). Interestingly, D1- and D2-neurons present distinct changes in activity after sucrose consumption that dynamically evolve throughout learning. During the aversive Pavlovian conditioning, D1- and D2-neurons present an increase in the activity in response to the CS and to the US (shock). Our data support a model in which D1- and D2-neurons are concurrently activated during appetitive and aversive conditioning.

Aversive conditioning increases short-term wariness but does not change habitat use in black bears associated with conflict.

Conflict between humans and black bears (Ursus americanus) occurs throughout North America with increasing public demand to replace lethal management with non-lethal methods, such as aversive conditioning (AC). AC aims to teach animals to associate negative stimuli with humans or their infrastructure. We sought to test the efficacy of AC using radio-collared black bears in Whistler, British Columbia, by monitoring individuals and assigning those in conflict with people to control or treatment groups. We measured wariness using overt reaction distance, displacement distance, and reaction to researchers before, during and after executing 3-5-day AC programs that consisted of launching projectiles at bears in the treatment group. We also assessed predictors of successful AC events (i.e., leaving at a run), changes in bear use of human-dominated habitat during the day and at night, and the effects of including a sound stimulus to signal the beginning and end of AC events. Among treated bears, overt reaction distance increased by 46.5% and displacement distance increased by 69.0% following AC programs, whereas both overt reaction distance and displacement distance decreased over time among control group bears. Each additional AC event during the previous 30 days increased likelihood of bear departure in response to researcher presence by 4.5%. The success of AC events varied among individuals, declined with distance to cover, and increased with exposure to previous AC events. Projectiles launched from guns were slightly more effective at causing bears to displace compared to those launched from slingshots, and sound stimuli decreased the likelihood of a successful AC event. AC did not alter diurnal use by bears of human-dominated habitat. Our results suggest that AC effectively increases short-term wariness in black bears but does not alter bear use of human-dominated spaces, highlighting the importance of proactive attractant management and prevention of food conditioning.

Aversive conditioning is impaired in impulsive individuals: A study on learning asymmetries

Background and objectivesAppetitive and aversive conditioning are thought to be involved in the development and maintenance of mental disorders including anxiety, mood, eating, and substance use disorders. However, few studies measure the relative strength of appetitive and aversive associations, and their relevance to the risk of mental disorders. This study aims to address this gap. MethodsWe tested how readily healthy volunteers acquire appetitive vs. aversive associations. 150 participants associated complex 3D objects with either gain or loss and made decisions to gain or avoid losing points. We investigated the relationship of a learning asymmetry with neuroticism, impulsivity, and anhedonia, to test the hypothesis that a stronger learning asymmetry corresponds to more extreme scores on these traits. ResultsImpulsivity was positively associated with the learning asymmetry (R2 = .10). This resulted from an inverse relation with the strength of aversive associations, indicating that impulsive individuals are worse at aversive learning. However, appetitive associations did not differ significantly. No correlations with neuroticism or anhedonia were found. LimitationsConditioning studies typically use primary reinforcers and a CS-. Lacking these may make these results less comparable to other studies. ConclusionsWe demonstrate that the learning asymmetry can measure individual differences linked to personality traits, and that impulsivity, normally linked with appetitive learning, also influences aversive learning. These results enable additional studies of learning asymmetry in relation to mental disorders, which could include measurements of mental health symptoms to provide further insight into how appetitive and aversive learning interacts with mental disorders.

Lipopolysaccharide (LPS) attenuates the primary conditioning of lithium chloride (LiCl)-induced context aversion but not the secondary conditioning of context aversion or taste avoidance

A first-order association can be formed between toxin-induced nausea and a context, as well as nausea and a taste cue. However, comparatively little is understood about second-order associations. The present study examined if the bacterial endotoxin, LPS, could impair the first- and second-order conditioning of context aversion (anticipatory nausea paradigm) and subsequent conditioned taste avoidance (two-bottle task). Adult male Long Evans rats were treated with LiCl (127 mg/kg, intraperitoneal [i.p.]) or vehicle control (NaCl) and then exposed to a distinct context for 4 first-order conditioning trials. LPS (200 μg/kg, i.p.) or NaCl were administered 24 h after each trial. Seventy-two h after the final first-order conditioning trial, rats underwent 2 second-order conditioning trials where they were treated with 2% saccharin (i.p.) and then exposed to the same context. Twenty-four h after the final second-order conditioning trial, rats were tested in a two-bottle task (2 trials), where they were given a choice between water and a palatable 0.2% saccharin solution. LiCl-treated rats demonstrated a context aversion by the 3rd conditioning trial in the anticipatory nausea paradigm. Rats previously exposed to LiCl also displayed a conditioned taste avoidance of saccharin within the two-bottle task. LPS attenuated first-order context aversion but did not alter either second-order context aversion or conditioned taste avoidance in the two-bottle task. This study demonstrated that a secondary association formed within an aversive context could result in a conditioned taste avoidance. Further, LPS may be able to attenuate primary conditioning, but not secondary conditioning.