A whole-brain analysis of functional connectivity and immediate early gene expression reveals functional network shifts after operant learning.

Previous studies of operant learning have addressed neuronal activities and network changes in specific brain areas, such as the striatum, sensorimotor cortex, prefrontal/orbitofrontal cortices, and hippocampus. However, how changes in the whole-brain network are caused by cellular-level changes remains unclear. We, therefore, combined resting-state functional magnetic resonance imaging (rsfMRI) and whole-brain immunohistochemical analysis of early growth response 1 (EGR1), a marker of neural plasticity, to elucidate the temporal and spatial changes in functional networks and underlying cellular processes during operant learning. We used an 11.7-Tesla MRI scanner and whole-brain immunohistochemical analysis of EGR1 in mice during the early and late stages of operant learning. In the operant training, mice received a reward when they pressed left and right buttons alternately, and were punished with a bright light when they made a mistake. A group of mice (n = 22) underwent the first rsfMRI acquisition before behavioral sessions, the second acquisition after 3 training-session-days (early stage), and the third after 21 training-session-days (late stage). Another group of mice (n = 40) was subjected to histological analysis 15 min after the early or late stages of behavioral sessions. Functional connectivity increased between the limbic areas and thalamus or auditory cortex after the early stage of training, and between the motor cortex, sensory cortex, and striatum after the late stage of training. The density of EGR1-immunopositive cells in the motor and sensory cortices increased in both the early and late stages of training, whereas the density in the amygdala increased only in the early stage of training. The subcortical networks centered around the limbic areas that emerged in the early stage have been implicated in rewards, pleasures, and fears. The connectivities between the motor cortex, somatosensory cortex, and striatum that consolidated in the late stage have been implicated in motor learning. Our multimodal longitudinal study successfully revealed temporal shifts in brain regions involved in behavioral learning together with the underlying cellular-level plasticity between these regions. Our study represents a first step towards establishing a new experimental paradigm that combines rsfMRI and immunohistochemistry to link macroscopic and microscopic mechanisms involved in learning.

Sex differences in neural representations of social and nonsocial reward in the medial prefrontal cortex.

The reinforcing nature of social interactions is necessary for the maintenance of appropriate social behavior. However, the neural substrates underlying social reward processing and how they might differ based on the sex and internal state of the animal remains unknown. It is also unclear whether these neural substrates are shared with those involved in nonsocial rewarding processing. We developed a fully automated, two choice (social-sucrose) operant assay in which mice choose between social and nonsocial rewards to directly compare the reward-related behaviors associated with two competing stimuli. We performed cellular resolution calcium imaging of medial prefrontal cortex (mPFC) neurons in male and female mice across varying states of water restriction and social isolation. We found that mPFC neurons maintain largely non-overlapping, flexible representations of social and nonsocial reward that vary with internal state in a sex-dependent manner. Additionally, optogenetic manipulation of mPFC activity during the reward period of the assay disrupted reward-seeking behavior across male and female mice. Thus, using a two choice operant assay, we have identified sex-dependent, non-overlapping neural representations of social and nonsocial reward in the mPFC that vary with internal state and that are essential for appropriate reward-seeking behavior.

High quality, high throughput, and low-cost simultaneous video recording of 60 animals in operant chambers using PiRATeMC.

BACKGROUND: The development of Raspberry Pi-based recording devices for video analyses of drug self-administration studies has been shown to be promising in terms of affordability, customizability, and capacity to extract in-depth behavioral patterns. Yet, most video recording systems are limited to a few cameras making them incompatible with large-scale studies. NEW METHOD: We expanded the PiRATeMC (Pi-based Remote Acquisition Technology for Motion Capture) recording system by increasing its scale, modifying its code, and adding equipment to accommodate large-scale video acquisition, accompanied by data on throughput capabilities, video fidelity, synchronicity of devices, and comparisons between Raspberry Pi 3B+ and 4B models. RESULTS: Using PiRATeMC default recording parameters resulted in minimal storage (∼350MB/h), high throughput (< ∼120 seconds/Pi), high video fidelity, and synchronicity within ∼0.02 seconds, affording the ability to simultaneously record 60 animals in individual self-administration chambers for various session lengths at a fraction of commercial costs. No consequential differences were found between Raspberry Pi models. COMPARISON WITH EXISTING METHOD(S): This system allows greater acquisition of video data simultaneously than other video recording systems by an order of magnitude with less storage needs and lower costs. Additionally, we report in-depth quantitative assessments of throughput, fidelity, and synchronicity, displaying real-time system capabilities. CONCLUSIONS: The system presented is able to be fully installed in a month's time by a single technician and provides a scalable, low cost, and quality-assured procedure with a high-degree of customization and synchronicity between recording devices, capable of recording a large number of subjects and timeframes with high turnover in a variety of species and settings.

Safety signals reinforce instrumental avoidance in humans.

Safety signals reinforce instrumental avoidance behavior in nonhuman animals. However, there are no conclusive demonstrations of this phenomenon in humans. Using human participants in an avoidance task, Experiments 1-3 and 5 were conducted online to assess the reinforcing properties of safety signals, and Experiment 4 was conducted in the laboratory. Participants were trained with CSs+ and CSs-, and they could avoid an aversive outcome during presentations of the CSs+ by pressing their space bar at a specific time. If successful, the aversive outcome was not presented but instead a safety signal was. Participants were then tested-whilst on extinction-with two new ambiguous test CSs. If participants made an avoidance response, one of the test CSs produced the trained safety signal and the other was a control. In Experiments 1 and 4, the control was followed by no signal. In Experiment 2, the control was followed by a signal that differed in one dimension (color) with the trained safety signal, and in Experiment 3, the control differed in two dimensions (shape and color) from the trained safety signal. Experiment 5 tested the reinforcing properties of the safety signal using a choice procedure and a new response during test. We observed that participants made more avoidance responses to the ambiguous test CSs when followed by the trained signal in Experiments 1, 3, 4, and 5 (but not in Experiment 2). Overall, these results suggest that trained safety signals can reinforce avoidance behavior in humans.

Kappa opioid receptor mediated operant performance in male and female rats.

Anhedonia and avolition are emotions frequently endorsed by individuals with stress related disorders. Kappa opioid receptor (KOR) activation can induce negative emotions and recent clinical evidence suggests that KOR antagonism can alleviate anhedonia in a transdiagnostic cohort of patients. However, the behavioral consequences of KOR activation and antagonism in modulating motivation, as assessed by schedule-controlled behavioral performance without preexisting conditions (stress or substance use), have not been formally assessed. To address this gap in the literature, this report utilized male and female Sprague Dawley rats to (1) evaluate the impact of the selective KOR agonist U50,488, on the performance of animals responding for sucrose pellets under a progressive ratio (PR) schedule and (2) determine the effects of the short-acting KOR antagonist LY2444296 alone and on U50,488 mediated reductions in PR performance. Overall, U50,488 5 mg/kg significantly reduced the breakpoint and number of rewards obtained by animals. This occurred in the absence of motor impairment and independent of evidence for satiation. LY2444296 did not alter PR performance when administered alone but effectively blocked the deficits induced by U50,488. To further delineate the behavioral alterations that underlie these reductions in responding, a more detailed analysis was conducted on PR performance in the first 15 min of the session, the period of time when animals obtained the most reinforcers. During this period, U50,488 increased the length of the post-reinforcement pause and reduced the running rate on PR schedules. These changes in behavior produced by acute activation of KORs are consistent with a reduction of effort-related motivation in rodents. These data contribute to the understanding of how KORs modulate motivation, which is critical to future efforts to evaluate performance in the context of stress and assess how KOR antagonists alleviate anhedonic behaviors associated with stress.

The effects of intra-accumbal administration of the nicotinic acetylcholine receptor agonist cytisine on the operant oral self-administration of ethanol were prevented by the GABAB receptor agonist baclofen in rats.

RATIONALE: Although the mesocorticolimbic dopamine (DA) system is the main neurochemical substrate that regulates the addictive and reinforcing effects of ethanol (EtOH), other neurotransmitter systems, such as the acetylcholine (Ach) system, modulate DAergic function in the nucleus accumbens (nAcc). Previously, we reported that intra-nAcc administration of the nicotinic Ach receptor agonist cytisine increased oral EtOH self-administration. GABAB receptors in the nAcc are expressed in DAergic terminals, inhibit the regulation of DA release into the nAcc, and could modulate the effects of cytisine on oral EtOH self-administration. The present study assessed the effects of intra-nAcc administration of the GABAB receptor agonist baclofen (BCF) on the impacts of cytisine on oral EtOH self-administration. METHODS: Male Wistar rats were deprived of water for 23.30 h and then trained to press a lever to receive EtOH on an FR3 schedule until a stable response rate of 80 % was achieved. After this training, the rats received an intra-nAcc injection of the nAch receptor agonist cytisine, BCF, and cytisine or 2-hydroxysaclofen, BCF, and cytisine before they were given access to EtOH on an FR3 schedule. RESULTS: Intra-nAcc injections of cytisine increased oral EtOH self-administration; this effect was reduced by BCF, and 2-hydroxysaclofen blocked the effects of BCF. CONCLUSIONS: These findings suggest that the reinforcing effects of EtOH are modulated not only by the DA system but also by other neurotransmitter systems involved in regulating DA release from DAergic terminals.

Negative allosteric modulation of CB1 cannabinoid receptor signalling decreases intravenous morphine self-administration and relapse in mice.

The endocannabinoid system interacts with the reward system to modulate responsiveness to natural reinforcers, as well as drugs of abuse. Previous preclinical studies suggested that direct blockade of CB1 cannabinoid receptors (CB1R) could be leveraged as a potential pharmacological approach to treat substance use disorder, but this strategy failed during clinical trials due to severe psychiatric side effects. Alternative strategies have emerged to circumvent the side effects of direct CB1 binding through the development of allosteric modulators. We hypothesized that negative allosteric modulation of CB1R signalling would reduce the reinforcing properties of morphine and decrease behaviours associated with opioid misuse. By employing intravenous self-administration in mice, we studied the effects of GAT358, a functionally-biased CB1R negative allosteric modulator (NAM), on morphine intake, relapse-like behaviour and motivation to work for morphine infusions. GAT358 reduced morphine infusion intake during the maintenance phase of morphine self-administration under a fixed ratio 1 schedule of reinforcement. GAT358 also decreased morphine-seeking behaviour after forced abstinence. Moreover, GAT358 dose dependently decreased the motivation to obtain morphine infusions under a progressive ratio schedule of reinforcement. Strikingly, GAT358 did not affect the motivation to work for food rewards in an identical progressive ratio task, suggesting that the effect of GAT358 in decreasing opioid self-administration was reward specific. Furthermore, GAT58 did not produce motor ataxia in the rotarod test. Our results suggest that CB1R NAMs reduced the reinforcing properties of morphine and could represent a viable therapeutic route to safely decrease misuse of opioids.

Goal-directed learning is multidimensional and accompanied by diverse and widespread changes in neocortical signaling.

New tasks are often learned in stages with each stage reflecting a different learning challenge. Accordingly, each learning stage is likely mediated by distinct neuronal processes. And yet, most rodent studies of the neuronal correlates of goal-directed learning focus on individual outcome measures and individual brain regions. Here, we longitudinally studied mice from naïve to expert performance in a head-fixed, operant conditioning whisker discrimination task. In addition to tracking the primary behavioral outcome of stimulus discrimination, we tracked and compared an array of object-based and temporal-based behavioral measures. These behavioral analyses identify multiple, partially overlapping learning stages in this task, consistent with initial response implementation, early stimulus-response generalization, and late response inhibition. To begin to understand the neuronal foundations of these learning processes, we performed widefield Ca2+ imaging of dorsal neocortex throughout learning and correlated behavioral measures with neuronal activity. We found distinct and widespread correlations between neocortical activation patterns and various behavioral measures. For example, improvements in sensory discrimination correlated with target stimulus evoked activations of response-related cortices along with distractor stimulus evoked global cortical suppression. Our study reveals multidimensional learning for a simple goal-directed learning task and generates hypotheses for the neuronal modulations underlying these various learning processes.

Keeping track of time: An interaction of mossy fibers and climbing fibers.

Precisely tracking time over second-long timescales is important for accurate anticipation and consequential actions, yet the neurobiological underpinnings remain unknown. In this issue of Neuron, Garcia-Garcia and colleagues1 show that computations in the cerebellum resulting from interactions between the mossy fiber and climbing fiber pathways contribute to long-interval learning during operant conditioning.

How to reduce fear in a snail: Take an aspirin, call me in the morning.

Aspirin (Acetylsalicylic acid, ASA), one of the widely used non-steroid anti-inflammatory drugs can easily end up in sewage effluents and thus it becomes necessary to investigate the effects of aspirin on behaviour of aquatic organisms. Previous studies in mammals have shown ASA to alter fear and anxiety-like behaviours. In the great pond snail Lymnaea stagnalis, ASA has been shown to block a 'sickness state' induced by lipopolysaccharide injection which upregulates immune and stress-related genes thus altering behavioural responses. In Lymnaea, eliciting physiological stress may enhance memory formation or block its retrieval depending on the stimulus type and intensity. Here we examine whether ASA will alter two forms of associative-learning memory in crayfish predator-experienced Lymnaea when ASA exposure accompanies predator-cue-induced stress during the learning procedure. The two trainings procedures are: 1) operant conditioning of aerial respiration; and 2) a higher form of learning, called configural learning, which here is dependent on evoking a fear response. We show here that ASA alone does not alter homeostatic aerial respiration, feeding behaviour or long-term memory (LTM) formation of operantly conditioned aerial respiration. However, ASA blocked the enhancement of LTM formation normally elicited by training snails in predator cue. ASA also blocked configural learning, which makes use of the fear response elicited by the predator cue. Thus, ASA alters how Lymnaea responds cognitively to predator detection.

Kv7 channel opener retigabine reduces self-administration of cocaine but not sucrose in rats.

The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug-seeking behaviours that can be modelled in rodents utilize the repeated intravenous self-administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that Kv7/KCNQ channels may contribute to the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter-delivered drug model systems, and the extent to which this effect generalizes to rats trained to self-administer drugs is not known. Here, we tested the ability of retigabine (ezogabine), a Kv7 channel opener, to regulate instrumental behaviour in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter-delivered cocaine in a conditioned place preference (CPP) assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine-SA under a fixed-ratio or progressive-ratio reinforcement schedule and found that retigabine pretreatment attenuated the SA of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self-administering sucrose, a natural reward. Compared with sucrose-SA, cocaine-SA was associated with reductions in the expression of the Kv7.5 subunit in the nucleus accumbens, without alterations in Kv7.2 and Kv7.3. Therefore, these studies reveal a reward-specific reduction in SA behaviour and support the notion that Kv7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.

Renewal of instrumental avoidance in humans.

The ABA renewal effect occurs when behavior is trained in one context (A), extinguished in a second context (B), and the test occurs in the training context (A). Two mechanisms that explain ABA renewal are context summation at the test and contextual modulation of extinction learning, with the former being unlikely if both contexts have a similar associative history. In two experiments, we used within-subjects designs in which participants learned to avoid a loud noise (unconditioned stimulus) signaled by discrete visual stimuli (conditioned stimuli [CSs]), by pressing the space bar on the computer keyboard. The training was conducted in two contexts, with a different pair of CSs (CS+ and CS-) trained in each context. During extinction, CS+ and CS- stimuli were presented in the alternative context from that of training, and participants were allowed to freely respond, but no loud noise was presented. Finally, all CSs were tested in both contexts, resulting in a within-subjects ABA versus ABB comparison. Across experiments, participants increased avoidance responses during training and decreased them during extinction, although Experiment 2 revealed less extinction. During the test, responding was higher when CS+ were tested in the training context (ABA) versus the extinction context (ABB), revealing the renewal of instrumental avoidance. Experiment 2 also measured expectancy after the avoidance test and revealed a remarkable similarity between avoidance responses and expectancy ratings. This study shows the renewal of instrumental avoidance in humans, and the results suggest the operation of a modulatory role for the context in renewal, similar to the occasion setting of extinction learning by the context. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Bird song comparison using deep learning trained from avian perceptual judgments.

Our understanding of bird song, a model system for animal communication and the neurobiology of learning, depends critically on making reliable, validated comparisons between the complex multidimensional syllables that are used in songs. However, most assessments of song similarity are based on human inspection of spectrograms, or computational methods developed from human intuitions. Using a novel automated operant conditioning system, we collected a large corpus of zebra finches' (Taeniopygia guttata) decisions about song syllable similarity. We use this dataset to compare and externally validate similarity algorithms in widely-used publicly available software (Raven, Sound Analysis Pro, Luscinia). Although these methods all perform better than chance, they do not closely emulate the avian assessments. We then introduce a novel deep learning method that can produce perceptual similarity judgements trained on such avian decisions. We find that this new method outperforms the established methods in accuracy and more closely approaches the avian assessments. Inconsistent (hence ambiguous) decisions are a common occurrence in animal behavioural data; we show that a modification of the deep learning training that accommodates these leads to the strongest performance. We argue this approach is the best way to validate methods to compare song similarity, that our dataset can be used to validate novel methods, and that the general approach can easily be extended to other species.

Category-specific general Pavlovian-instrumental transfer.

Modern living is characterized by easy access to highly palatable energy-dense foods. Environmental cues associated with palatable foods increase seeking of those foods (specific transfer) and other palatable foods (general transfer). We conducted a series of studies testing the boundaries of food cue-reactivity by evaluating the impact of broader flavor associations (i.e. saltiness, sweetness) in eliciting general transfer effects. Experiment 1 was an online experiment with fictive rewards that tested if two actions associated with different food rewards (chip and chocolate points) could be provoked by images of other foods that were either similar or distinct in flavor from the foods associated with these instrumental actions. We observed that response excitation was only elicited by similarly flavored food cues, whereas distinctly flavored food cues inhibited response rates relative to control cues. Experiment 2 confirmed this observation in a classroom setting where real food rewards were contingent on task performance. Experiment 3 was an online study that further confirmed the reliability of the effects with a well powered sample. There were moderate-to-strong associations between specific and general transfer effects across all studies, suggesting overlapping cognitive processes are responsible for both transfer effects. These data improve the mechanistic understanding of how broad category associations can moderate the impact of food cues on food choices. This knowledge could be helpful for improving the precision of psychological interventions that seek to mitigate the impact of food cue-reactivity.

Manipulations of the context-response relationship reduce the expression of response habits.

Habitual instrumental behaviour is believed to rely on stimulus-response (S-R) associations. However, the method most commonly used to identify habitual behaviour, outcome devaluation, provides only indirect evidence of S-R control. Therefore, it is important to have a better understanding of the S-R association believed to underlie habitual responding. Under free-operant conditions, the context itself likely serves as at least part of the relevant stimuli in the association, and so modifications to the predictive power of the context should alter the expression of habits. The following experiments investigated how changes to the relationship between the training context and performance of the response, either by changing the context during testing or by exposing animals to the context alone, without the response lever present, impacted behavioural control during a devaluation test. We found evidence that the training context is important for the expression of habits; testing animals in a different context than where they were trained resulted in increased goal-directed control (Experiment 1). Furthermore, context alone exposure also increased goal-directed control with animals that received context alone exposure showing stronger devaluation effects, whether the context alone exposure happened on the last day of training (Experiment 2) or throughout training (Experiment 3). These findings are consistent with prior reports that the training context is important for the expression of habits and extends these findings by using sensory-specific satiety as a means for devaluation and by using context alone exposure to alter behavioural control.

Connecting self-report and instrumental behavior during incubation of food craving in humans.

Incubation of craving is a phenomenon describing the intensification of craving for a reward over extended periods of abstinence from reinforcement. Animal models use instrumental markers of craving to reward cues to examine incubation, while human paradigms rely on subjective self-reports. Here, we characterize an animal-inspired, novel human paradigm that showed strong positive relationships between self-reports and instrumental markers of craving for favored palatable foods. Further, we found consistent nonlinear relationships with time since last consumption and self-reports, and preliminary patterns between time and instrumental responses. These findings provide a novel approach to establishing an animal-inspired human model of incubation.

Random interval schedule of reinforcement influences punishment resistance for cocaine in rats.

In an animal model of compulsive drug use, a subset of rats continues to self-administer cocaine despite footshock consequences and is considered punishment resistant. We recently found that punishment resistance is associated with habits that persist under conditions that typically encourage a transition to goal-directed control. Given that random ratio (RR) and random interval (RI) schedules of reinforcement influence whether responding is goal-directed or habitual, we investigated the influence of these schedules on punishment resistance for cocaine or food. Male and female Sprague Dawley rats were trained to self-administer either intravenous cocaine or food pellets on a seeking-taking chained schedule of reinforcement, with the seeking lever requiring completion of either an RR20 or RI60 schedule. Rats were then given four days of punishment testing with footshock administered at the completion of seeking on a random one-third of trials. For cocaine-trained rats, the RI60 schedule led to greater punishment resistance (i.e., more trials completed) than the RR20 schedule in males and females. For food-trained rats, the RI60 schedule led to greater punishment resistance (i.e., higher reward rates) than the RR20 schedule in female rats, although male rats showed punishment resistance on both RR20 and RI60 schedules. For both cocaine and food, we found that seeking responses were suppressed to a greater degree than reward rate with the RI60 schedule, whereas response rate and reward rate were equally suppressed with the RR20 schedule. This dissociation between punishment effects on reward rate and response rate with the RI60 schedule can be explained by the nonlinear relation between these variables on RI schedules, but it does not account for the enhanced resistance to punishment. Overall, the results show greater punishment resistance with the RI60 schedule as compared to the RR20 schedule, indicating that schedules of reinforcement are an influencing factor on resistance to negative consequences.

Recording Neural Reward Signals in a Naturalistic Operant Task Using Mobile-EEG and Augmented Reality.

The electrophysiological response to rewards recorded during laboratory tasks has been well documented, yet little is known about the neural response patterns in a more naturalistic setting. Here, we combined a mobile-EEG system with an augmented reality headset to record event-related brain potentials (ERPs) while participants engaged in a naturalistic operant task to find rewards. Twenty-five participants were asked to navigate toward a west or east goal location marked by floating orbs, and once participants reached the goal location, the orb would then signify a reward (5 cents) or no-reward (0 cents) outcome. Following the outcome, participants returned to a start location marked by floating purple rings, and once standing in the middle, a 3 s counter signaled the next trial, for a total of 200 trials. Consistent with previous research, reward feedback evoked the reward positivity, an ERP component believed to index the sensitivity of the anterior cingulate cortex to reward prediction error signals. The reward positivity peaked ∼230 ms with a maximal at channel FCz (M = -0.695 µV, ±0.23) and was significantly different than zero (p < 0.01). Participants took ∼3.38 s to reach the goal location and exhibited a general lose-shift (68.3% ±3.5) response strategy and posterror slowing. Overall, these novel findings provide support for the idea that combining mobile-EEG with augmented reality technology is a feasible solution to enhance the ecological validity of human electrophysiological studies of goal-directed behavior and a step toward a new era of human cognitive neuroscience research that blurs the line between laboratory and reality.

Resurgence mitigation across extended extinction following four and eight cycles of on/off alternative reinforcement.

Resurgence is an increase in an extinguished operant response resulting from a worsening of conditions (e.g., extinction) for a more recently reinforced alternative behavior. Previous research has shown that exposure to cycles of alternative reinforcement available versus unavailable (i.e., on/off alternative reinforcement) across sessions can reduce subsequent resurgence. Most previous assessments of the procedure have examined target operant responding during only single-session resurgence tests, and it remains unclear if exposure to relatively few cycles of on/off alternative reinforcement can maintain low rates of target behavior across extended exposure to extinction. This experiment with rats examined the effects of 4 or 8 cycles of on/off alternative reinforcement on subsequent resurgence during a 10-session extinction test. The results show that exposure to 4 cycles of on/off alternative reinforcement is as effective as 8 cycles in producing low rates of target behavior during treatment and across extended extinction. This result is consistent with extant theories of resurgence and suggests that on/off alternative reinforcement could have translational utility following relatively few cycles of exposure.

Unraveling the influence of Pavlovian cues on decision-making: A pre-registered meta-analysis on Pavlovian-to-instrumental transfer.

Amidst the replicability crisis, promoting transparency and rigor in research becomes imperative. The Pavlovian-to-Instrumental Transfer (PIT) paradigm is increasingly used in human studies to offer insights into how Pavlovian cues, by anticipating rewards or punishments, influence decision-making and potentially contribute to the development of clinical conditions. However, research on this topic faces challenges, including methodological variability and the need for standardized approaches, which can undermine the quality and robustness of experimental findings. Hence, we conducted a meta-analysis to unravel the methodological, task-related, individual, training, and learning factors that can modulate PIT. By scrutinizing these factors, the present meta-analysis reviews the current literature on human PIT, provides practical guidelines for future research to enhance study outcomes and refine methodologies, and identifies knowledge gaps that can serve as a direction for future studies aiming to advance the comprehension of how Pavlovian cues shape decision-making.