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.

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.

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.

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.

The ventral tegmental area dopamine to basolateral amygdala projection supports acquisition of cocaine self-administration.

Dopamine signaling in the amygdala is known to play a role in associative learning and memory, including the process of learning to associate environmental cues with the reinforcing properties of drugs like cocaine. Evidence suggests that the ventral tegmental area (VTA) dopamine (DA) projection specifically to the basolateral amygdala (BLA) participates in establishing cocaine-cue associations that can promote later craving- and relapse-like responses to the cue alone. In order to further investigate the specific role of VTA-BLA projections in cocaine-reinforced learning, we used chemogenetics to manipulate VTA DA inputs to the BLA during cocaine self-administration, cue- and cocaine-primed reinstatement, and conditioned place preference. We found inhibiting DA input to the BLA during cocaine self-administration inhibited acquisition and weakened the ability of the previously cocaine-paired cue to elicit cocaine-seeking, while acutely inhibiting the pathway on the day of cue-induced reinstatement testing had no effect. Conversely, exciting the projection during self-administration boosted the salience of the cocaine-paired cue as indicated by enhanced responding during cue-induced reinstatement. Importantly, interfering with DA input to the BLA had no impact on the ability of cocaine to elicit a place preference or induce reinstatement in response to a priming cocaine injection. Overall, we show that manipulation of projections underlying DA signaling in the BLA may be useful for developing therapeutic interventions for substance use disorders.

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.

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.

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.

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).

Self-administration acquisition latency predicts locomotor sensitivity to cocaine in male rats.

Individual differences in drug use emerge soon after initial exposure, and only a fraction of individuals who initiate drug use go on to develop a substance use disorder. Variability in vulnerability to establishing drug self-administration behavior is also evident in preclinical rodent models. Latent characteristics that underlie this variability and the relationship between early drug use patterns and later use remain unclear. Here, we attempt to determine whether propensity to establish cocaine self-administration is related to subsequent cocaine self-administration behavior in male Sprague-Dawley rats (n = 14). Prior to initiating training, we evaluated basal locomotor and anxiety-like behavior in a novel open field test. We then trained rats to self-administer cocaine in daily 3 h cocaine (0.75 mg/kg/infusion) self-administration sessions until acquisition criteria (≥30 active lever presses with ≥70 % responding on the active lever in one session) was met and divided rats into Early and Late groups by median-split analysis based on their latency to meet acquisition criteria. After each rat met acquisition criteria, we gave them 10 additional daily cocaine self-administration sessions. We then conducted a progressive ratio, cocaine-induced locomotor sensitivity test, and non-reinforced cocaine seeking test after two weeks of forced abstinence. Early Learners exhibited significantly less locomotion after an acute injection of cocaine, but the groups did not differ in any other behavioral parameter examined. These results indicate that cocaine self-administration acquisition latency is not predictive of subsequent drug-taking behavior, but may be linked to physiological factors like drug sensitivity that can predispose rats to learn the operant task.

Conditional deletion of the AMPA-GluA1 and NMDA-GluN1 receptor subunit genes in midbrain D1 neurons does not alter cocaine reward in mice.

Synaptic plasticity in the mesolimbic dopamine (DA) system contributes to the neural adaptations underlying addictive behaviors and relapse. However, the specific behavioral relevance of glutamatergic excitatory drive onto dopamine D1 receptor (D1R)-expressing neurons in mediating the reinforcing effect of cocaine remains unclear. Here, we investigated how midbrain AMPAR and NMDAR function modulate cocaine reward-related behavior using mutant mouse lines lacking the glutamate receptor genes Gria1 or Grin1 in D1R-expressing neurons (GluA1D1CreERT2 or GluN1D1CreERT2, respectively). We found that conditional genetic deletion of either GluA1 or GluN1 within this neuronal sub-population did not impact the ability of acute cocaine injection to increase intracranial self-stimulation (ICSS) ratio or reduced brain reward threshold compared to littermate controls. Additionally, our data demonstrate that deletion of GluA1 and GluN1 receptor subunits within D1R-expressing neurons did not affect cocaine reinforcement in an operant self-administration paradigm, as mutant mice showed comparable cocaine responses and intake to controls. Given the pivotal role of glutamate receptors in mediating relapse behavior, we further explored the impact of genetic deletion of AMPAR and NMDAR onto D1R-expressing neurons on cue-induced reinstatement following extinction. Surprisingly, deletion of AMPAR and NMDAR onto these neurons did not impair cue-induced reinstatement of cocaine-seeking behavior. These findings suggest that glutamatergic activity via NMDAR and AMPAR in D1R-expressing neurons may not exclusively mediate the reinforcing effects of cocaine and cue-induced reinstatement.

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.

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.

Compulsive avoidance in youths and adults with OCD: an aversive pavlovian-to-instrumental transfer study.

Compulsive behaviour may often be triggered by Pavlovian cues. Assessing how Pavlovian cues drive instrumental behaviour in obsessive-compulsive disorder (OCD) is therefore crucial to understand how compulsions develop and are maintained. An aversive Pavlovian-to-Instrumental transfer (PIT) paradigm, particularly one involving avoidance/cancellation of negative outcomes, can enable such investigation and has not previously been studied in clinical-OCD. Forty-one participants diagnosed with OCD (21 adults; 20 youths) and 44 controls (21 adults; 23 youths) completed an aversive PIT task. Participants had to prevent the delivery of unpleasant noises by moving a joystick in the correct direction. They could infer these correct responses by learning appropriate response-outcome (instrumental) and stimulus-outcome (Pavlovian) associations. We then assessed whether Pavlovian cues elicited specific instrumental avoidance responses (specific PIT) and induced general instrumental avoidance (general PIT). We investigated whether task learning and confidence indices influenced PIT strength differentially between groups. There was no overall group difference in PIT performance, although youths with OCD showed weaker specific PIT than youth controls. However, urge to avoid unpleasant noises and preference for safe over unsafe stimuli influenced specific and general PIT respectively in OCD, while PIT in controls was more influenced by confidence in instrumental and Pavlovian learning. Thus, in OCD, implicit motivational factors, but not learnt knowledge, may contribute to the successful integration of aversive Pavlovian and instrumental cues. This implies that compulsive avoidance may be driven by these automatic processes. Youths with OCD show deficits in specific PIT, suggesting cue integration impairments are only apparent in adolescence. These findings may be clinically relevant as they emphasise the importance of targeting such implicit motivational processes when treating OCD.

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.

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.

VTA glutamatergic projections to the nucleus accumbens suppress psychostimulant-seeking behavior.

Converging evidence indicates that both dopamine and glutamate neurotransmission within the nucleus accumbens (NAc) play a role in psychostimulant self-administration and relapse in rodent models. Increased NAc dopamine release from ventral tegmental area (VTA) inputs is critical to psychostimulant self-administration and NAc glutamate release from prelimbic prefrontal cortex (PFC) inputs synapsing on medium spiny neurons (MSNs) is critical to reinstatement of psychostimulant-seeking after extinction. The regulation of the activity of MSNs by VTA dopamine inputs has been extensively studied, and recent findings have demonstrated that VTA glutamate neurons target the NAc medial shell. Here, we determined whether the mesoaccumbal glutamatergic pathway plays a role in psychostimulant conditioned place preference and self-administration in mice. We used optogenetics to induce NAc release of glutamate from VTA inputs during the acquisition, expression, and reinstatement phases of cocaine- or methamphetamine-induced conditioned place preference (CPP), and during priming-induced reinstatement of cocaine-seeking behavior. We found that NAc medial shell release of glutamate resulting from the activation of VTA glutamatergic fibers did not affect the acquisition of cocaine-induced CPP, but it blocked the expression, stress- and priming-induced reinstatement of cocaine- and methamphetamine CPP, as well as it blocked the priming-induced reinstatement of cocaine-seeking behavior after extinction. These findings indicate that in contrast to the well-recognized mesoaccumbal dopamine system that is critical to psychostimulant reward and relapse, there is a parallel mesoaccumbal glutamatergic system that suppresses reward and psychostimulant-seeking behavior.

Two Distinct Neuronal Populations in the Rat Parafascicular Nucleus Oppositely Encode the Engagement in Stimulus-driven Reward-seeking.

BACKGROUND: The thalamus is a phylogenetically well-preserved structure. Known to densely contact cortical regions, its role in the transmission of sensory information to the striatal complex has been widely reconsidered in recent years. METHODS: The parafascicular nucleus of the thalamus (Pf) has been implicated in the orientation of attention toward salient sensory stimuli. In a stimulus-driven reward-seeking task, we sought to characterize the electrophysiological activity of Pf neurons in rats. RESULTS: We observed a predominance of excitatory over inhibitory responses for all events in the task. Neurons responded more strongly to the stimulus compared to lever-pressing and reward collecting, confirming the strong involvement of the Pf in sensory information processing. The use of long sessions allowed us to compare neuronal responses to stimuli between trials when animals were engaged in action and those when they were not. We distinguished two populations of neurons with opposite responses: MOTIV+ neurons responded more intensely to stimuli followed by a behavioral response than those that were not. Conversely, MOTIV- neurons responded more strongly when the animal did not respond to the stimulus. In addition, the latency of excitation of MOTIV- neurons was shorter than that of MOTIV+ neurons. CONCLUSION: Through this encoding, the Pf could perform an early selection of environmental stimuli transmitted to the striatum according to motivational level.

From land to water: "Sunken" T-maze for associated learning in cichlid fish.

The study introduced and evaluated learning paradigms for Maylandia callainos cichlids using a modified version of the rodent T-maze, filled with tank water (the "sunken" modification). Both male and female fish underwent training in two distinct conditioning paradigms. Firstly, simple operant conditioning involved placing a food reward in either the right or left compartment. Cichlids demonstrated the ability to purposefully find the bait within 6 days of training, with a persistent place preference lasting up to 6 days. Additionally, the learning dynamics varied with sex: female cichlids exhibited reduction in latency to visit the target compartment and consume the bait, along with a decrease in the number of errors 3 and 4 days earlier than males, respectively. Secondly, visually-cued operant conditioning was conducted, with a food reward exclusively placed in the yellow compartment, randomly positioned on the left or right side of the maze during each training session. Visual learning persisted for 10 days until reaction time improvement plateaued. Color preference disappeared after 4 consecutive check-ups, with no sex-related interference. For further validation of visually-cued operant conditioning paradigm, drugs MK-801 (dizocilpine) and caffeine, known to affect performance in learning tasks, were administered intraperitoneally. Chronic MK-801 (0.17 mg/kg) impaired maze learning, resulting in no color preference development. Conversely, caffeine administration enhanced test performance, increasing precision in fish. This developed paradigm offers a viable approach for studying learning and memory and presents an effective alternative to rodent-based drug screening tools, exhibiting good face and predictive validity.