Habituation: It's not what you think it is.

In this review, we take a critical look at the methods used to document habituation and the theoretical assumptions that have been made about it. We point out problems associated with measuring habituation merely as a change over the course of repeated presentations of a stimulus. We argue that a common test procedure is essential to assess the relative magnitudes of habituation learning especially when different training procedures are examined. We further suggest that this would be required in order to draw meaningful conclusions about the conditions for optimizing habituation. We also challenge the view that habituation is nonassociative and consider the implications of various associative learning perspectives not only for context-specific habituation but for encoding a representation of the stimulus. We conclude with our recommendations for future research on habituation and we highlight the need to integrate behavioral and neurobiological studies.

Developments in associative theory: A tribute to the contributions of Robert A. Rescorla.

The field of associative learning theory was forever changed by the contributions of Robert A. Rescorla. He created an organizational structure that gave us a framework for thinking about the key questions surrounding learning theory: what are the conditions that produce learning?, what is the content of that learning?, and how is that learning expressed in performance? He gave us beautifully sophisticated experimental designs that tackled deep theoretical problems in experimentally clever and elegant ways. And he left us with a collection of work that fundamentally altered the way we as a field think about basic learning processes. Few scientists have impacted their field in the way that Rescorla impacted animal learning theory. In this paper, we introduce this special issue (Developments in Associative Theory: A Tribute to Robert A. Rescorla) by considering some of the many ways in which Rescorla's empirical and theoretical contributions impacted learning theory over his almost 50-year career. We conclude by identifying multiple fundamental issues we think he would have found especially fruitful to pursue as we continue to move forward. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Analysis of vertebrate vision in a 384-well imaging system.

Visual impairment affects 253 million people worldwide and new approaches for prevention and treatment are urgently needed. While small molecules with potential beneficial effects can be examined in various model systems, the in vivo evaluation of visual function remains a challenge. The current study introduces a novel imaging system for measuring visually-guided behaviors in larval zebrafish. The imaging system is the first to image four 96-well plates with a single camera for automated measurements of activity in a 384-well format. In addition, it is the first system to project moving visual stimuli and analyze the optomotor response in the wells of a 96-well plate. We found that activity is affected by tricaine, diazepam and flumazenil. Surprisingly, diazepam treatments induce a loss of visual responses, at concentrations that do not affect activity or induce hyperactivity. Overall, our studies show that the developed imaging system is suitable for automated measurements of vertebrate vision in a high-throughput format.

Behavioral studies of stimulus learning in zebrafish larvae.

Within a week of fertilization, a zebrafish larva has developed a robust behavioral repertoire that includes the ability to learn about noncontingent stimuli. I begin this paper with a brief review of the t1-t2 framework in which groups receive different experiences at the first time point (t1) followed by a common assessment at the second time point (t2) and the strengths of this framework for studying stimulus learning. I then describe assays that have been implemented within the t1-t2 framework to demonstrate stimulus learning in the developing zebrafish. I discuss how these assays have been used to address three fundamental questions about stimulus learning: What are the conditions for stimulus learning? What is the content of stimulus learning? How is stimulus learning reflected in behavior? For each of these three questions, I also identify those issues regarding stimulus learning in the developing zebrafish that warrant further analysis at the behavioral level.

The loss and recovery of vertebrate vision examined in microplates.

Regenerative medicine offers potentially ground-breaking treatments of blindness and low vision. However, as new methodologies are developed, a critical question will need to be addressed: how do we monitor in vivo for functional success? In the present study, we developed novel behavioral assays to examine vision in a vertebrate model system. In the assays, zebrafish larvae are imaged in multiwell or multilane plates while various red, green, blue, yellow or cyan objects are presented to the larvae on a computer screen. The assays were used to examine a loss of vision at 4 or 5 days post-fertilization and a gradual recovery of vision in subsequent days. The developed assays are the first to measure the loss and recovery of vertebrate vision in microplates and provide an efficient platform to evaluate novel treatments of visual impairment.

Effects of embryonic exposure to polychlorinated biphenyls (PCBs) on anxiety-related behaviors in larval zebrafish.

The zebrafish (Danio rerio) is an excellent model system for assessing the effects of toxicant exposure on behavior and neurodevelopment. In the present study, we examined the effects of sub-chronic embryonic exposure to polychlorinated biphenyls (PCBs), a ubiquitous anthropogenic pollutant, on anxiety-related behaviors. We found that exposure to the PCB mixture, Aroclor (A) 1254, from 2 to 26h post-fertilization (hpf) induced two statistically significant behavioral defects in larvae at 7 days post-fertilization (dpf). First, during 135min of free swimming, larvae that had been exposed to 2ppm, 5ppm or 10ppm A1254 exhibited enhanced thigmotaxis (edge preference) relative to control larvae. Second, during the immediately ensuing 15-min visual startle assay, the 5ppm and 10ppm PCB-exposed larvae reacted differently to a visual threat, a red 'bouncing' disk, relative to control larvae. These results are consistent with the anxiogenic and attention-disrupting effects of PCB exposure documented in children, monkeys and rodents and merit further investigation.

Effects of embryonic exposure to polychlorinated biphenyls (PCBs) on larval zebrafish behavior.

Developmental disorders such as anxiety, autism, and attention deficit hyperactivity disorders have been linked to exposure to polychlorinated biphenyls (PCBs), a ubiquitous anthropogenic pollutant. The zebrafish is widely recognized as an excellent model system for assessing the effects of toxicant exposure on behavior and neurodevelopment. In the present study, we examined the effect of sub-chronic embryonic exposure to the PCB mixture, Aroclor (A) 1254 on anxiety-related behaviors in zebrafish larvae at 7 days post-fertilization (dpf). We found that exposure to low concentrations of A1254, from 2 to 26 h post-fertilization (hpf) induced specific behavioral defects in two assays. In one assay with intermittent presentations of a moving visual stimulus, 5 ppm and 10 ppm PCB-exposed larvae displayed decreased avoidance behavior but no significant differences in thigmotaxis or freezing relative to controls. In the other assay with intermittent presentations of a moving visual stimulus and a stationary visual stimulus, 5 ppm and 10 ppm PCB-exposed larvae had elevated baseline levels of thigmotaxis but no significant differences in avoidance behavior relative to controls. The 5 ppm larvae also displayed higher terminal levels of freezing relative to controls. Collectively, our results show that exposure to ecologically valid PCB concentrations during embryonic development can induce functional deficits and alter behavioral responses to a visual threat.

Brief and extended alcohol-cue-exposure effects on craving and attentional bias.

Past research has shown that underage college-student drinkers (UCSDs) report increased subjective craving and exhibit stronger attentional biases to alcohol following alcohol-cue exposure. To date, less research has examined whether momentary decreases in alcohol craving are associated with reductions in attentional bias. One experimental manipulation that has been used to produce within-session decreases in alcohol craving is to extend the duration of laboratory-based alcohol-cue exposure protocols. The aim of this study was to examine the effects of both brief and extended alcohol-cue exposure on subjective craving and attentional bias among UCSDs. Eighty participants were randomized either to a group that received a short, in vivo, alcohol-cue-exposure period (short-exposure group [SE], 2 3-min blocks) or to a group that received a long-exposure period (long-exposure group [LE], 6 3-min blocks). Both groups completed a visual probe task before and after cue exposure to assess changes in attentional bias. Analyses revealed no group differences in mean craving or mean attentional bias before or after cue exposure. Further, exploratory analyses revealed no sex differences in our measures of craving or attentional bias. For Group LE, but not Group SE, within-session changes in craving positively predicted within-session changes in attentional bias. However, further analyses revealed that this relationship was significant only for women in the LE group. Implications for treatments that aim to reduce craving and/or attentional bias are discussed.

Effects of embryonic cyclosporine exposures on brain development and behavior.

Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures.

Alcohol-cue exposure effects on craving and attentional bias in underage college-student drinkers.

The effect of alcohol-cue exposure on eliciting craving has been well documented, and numerous theoretical models assert that craving is a clinically significant construct central to the motivation and maintenance of alcohol-seeking behavior. Furthermore, some theories propose a relationship between craving and attention, such that cue-induced increases in craving bias attention toward alcohol cues, which, in turn, perpetuates craving. This study examined the extent to which alcohol cues induce craving and bias attention toward alcohol cues among underage college-student drinkers. We designed within-subject cue-reactivity and visual-probe tasks to assess in vivo alcohol-cue exposure effects on craving and attentional bias on 39 undergraduate college drinkers (ages 18-20). Participants expressed greater subjective craving to drink alcohol following in vivo cue exposure to a commonly consumed beer compared with water exposure. Furthermore, following alcohol-cue exposure, participants exhibited greater attentional biases toward alcohol cues as measured by a visual-probe task. In addition to the cue-exposure effects on craving and attentional bias, within-subject differences in craving across sessions marginally predicted within-subject differences in attentional bias. Implications for both theory and practice are discussed. (PsycINFO Database Record

High-throughput analysis of behavior in zebrafish larvae: effects of feeding.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. Identification of these factors is facilitated by high-throughput analyses of behavior in zebrafish larvae, which can be imaged in multiwell or multilane plates. However, the nutritional needs of zebrafish larvae during the behavioral experiments are not fully understood. Zebrafish larvae begin feeding between 4 and 5 days postfertilization (dpf), but can live solely on nutrients derived from the yolk until at least 7 dpf. To examine whether feeding affects behavior, we measured a broad range of behaviors with and without feeding at 5, 6, and 7 dpf. We found that feeding did not have a significant effect on behavior in 5-day-old larvae. In contrast, fed 6- and 7-day-old larvae displayed increased avoidance responses to visual stimuli, increased swim speeds, and decreased resting in comparison to unfed larvae. In addition, the fed 7-day-old larvae displayed a decrease in thigmotaxis and a decrease in the distance between larvae in the presence of visual stimuli. Thus, feeding affects a range of behaviors in 6- and 7-day-old larvae. We conclude that 5-day-old larvae are well-suited for high-throughput analyses of behavior, since effects of feeding can be avoided at this time. For high-throughput analyses of behavior in older larvae, standard feeding protocols need to be developed.

Single stimulus learning in zebrafish larvae.

Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7dpf larvae and extended the finding to 5 and 6dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed.

Pavlovian contextual and instrumental biconditional discrimination learning in mice.

Genetically-modified animal models are a powerful tool for investigating the link between neurological and behavioral changes and for the development of therapeutic interventions. Executive function deficits are symptomatic of many human clinical disorders but few tasks exist for studying executive functions in mice. To address this need, we describe procedures for establishing Pavlovian contextual and instrumental biconditional discriminations (BCDs) in C57BL/6J mice. In the first experiment, contextual cues disambiguated when two short duration stimulus targets would be followed by food pellets. In the second experiment, discrete visual cues signaled when lever press or nose poke responses would be continuously reinforced with food pellets. Mice learned both BCDs as evidenced by differential responding in each cue during training and, more critically, during extinction testing. The implications of these findings for using BCD tasks to analyze the neural substrates of executive processing in animal models are discussed.

Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.

Neurobehavioral disorders such as anxiety, autism, and attention deficit hyperactivity disorders are typically influenced by genetic and environmental factors. Although several genetic risk factors have been identified in recent years, little is known about the environmental factors that either cause neurobehavioral disorders or contribute to their progression in genetically predisposed individuals. One environmental factor that has raised concerns is chlorpyrifos, an organophosphate pesticide that is widely used in agriculture and is found ubiquitously in the environment. In the present study, we examined the effects of sub-chronic chlorpyrifos exposure on anxiety-related behavior during development using zebrafish larvae. We found that sub-chronic exposure to 0.01 or 0.1 µM chlorpyrifos during development induces specific behavioral defects in 7-day-old zebrafish larvae. The larvae displayed decreases in swim speed and thigmotaxis, yet no changes in avoidance behavior were seen. Exposure to 0.001 µM chlorpyrifos did not affect swimming, thigmotaxis, or avoidance behavior and exposure to 1 µM chlorpyrifos induced behavioral defects, but also induced defects in larval morphology. Since thigmotaxis, a preference for the edge, is an anxiety-related behavior in zebrafish larvae, we propose that sub-chronic chlorpyrifos exposure interferes with the development of anxiety-related behaviors. The results of this study provide a good starting point for examination of the molecular, cellular, developmental, and neural mechanisms that are affected by environmentally relevant concentrations of organophosphate pesticides. A more detailed understanding of these mechanisms is important for the development of predictive models and refined health policies to prevent toxicant-induced neurobehavioral disorders.

Pavlovian biconditional discrimination learning in the C57BL/6J mouse.

Four experiments using mice examined acquisition of Pavlovian biconditional discriminations in which two stimulus compounds were paired with food (AX+ and BY+) and two were not (AY- and BX-). Temporally asynchronous compounds were generated by using contextual stimuli (Experiment 1) and 15-s discrete visual cues (Experiments 2A, 2B and 3) to disambiguate when embedded noise or tone stimuli would be paired with food. When food pellets followed both reinforced compounds, successful acquisition was obtained in Experiment 1 but not in Experiments 2A and 2B even though the order of trials was modeled after that used in Experiment 1. However, when differential outcomes followed the reinforced compounds in Experiment 3, acquisition was obtained with discrete cue stimulus compounds. The implications of these results for modulatory models of conditional discrimination learning in animals are discussed.

Imaging escape and avoidance behavior in zebrafish larvae.

This review provides an overview of the assays that are used for measuring escape and avoidance behavior in zebrafish, with a specific focus on zebrafish larvae during the first week of development. Zebrafish larvae display a startle response when exposed to tactile, acoustic, or visual stimuli and will avoid dark areas, moving objects, conspecifics, and open spaces. Emotional states such as fear and anxiety might be induced when larvae are exposed to stimuli that they would normally escape from or avoid. Although these emotional states probably differ between species and change during development, much can be learned about human fear and anxiety using zebrafish as a model system. The molecular mechanisms of fear and anxiety are highly conserved in vertebrates and are present during early zebrafish development. Larvae during the first week of development display elevated cortisol levels in response to stress and are sensitive to the same anxiolytics that are used for the management of anxiety in humans. Zebrafish larvae are well suited for high-throughput analyses of behavior, and automated systems have been developed for imaging and analyzing the behavior of zebrafish larvae in multiwell plates. These high-throughput analyses will not only provide a wealth of information on the genes and environmental factors that influence escape and avoidance behaviors and the emotional states that might accompany them but will also facilitate the discovery of novel pharmaceuticals that could be used in the management of anxiety disorders in humans.

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders.

Locomotor behaviors in zebrafish (Danio rerio) larvae.

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.

Effect of the passage of time on the contribution of initial response-outcome associations to instrumental performance.

The effect of the passage of time on the contribution of initial response-outcome associations to subsequent instrumental performance was explored in three experiments with rats using outcome devaluation. Experiments 1 and 2 showed that a response that had been trained first with one outcome and then given identical training with a second outcome was more sensitive to devaluation of the second outcome than the first if the two training episodes were separated in time. Experiment 3 showed that inserting a delay between training with the second outcome and testing after outcome devaluation appeared to mitigate this effect of temporally separating first and second outcome training. Inserting this delay also made a response slightly more sensitive to devaluation of the first outcome than the second when there was no delay between the two training episodes. These results suggest that the passage of time can shift the balance between the contributions of first and second trained outcomes to instrumental performance.

Effect of US identity on elimination and recovery of autoshaped responding with explicitly unpaired and degraded contingency extinction procedures.

The depressive effects of noncontingent and explicitly unpaired food unconditioned stimuli (USs) and the recovery from those effects on autoshaped responding were examined in a series of experiments with pigeons. In Experiments 1 and 2, responding to a keylight conditioned stimulus (CS) previously paired with food was depressed equally by explicitly unpaired presentations of either that same food or a different food. Furthermore, responding recovered equally following removal of the explicitly unpaired foods. In contrast, Experiments 3 and 4 showed that noncontingent presentations of a food US depressed responding more to a keylight CS paired with that same food than to a keylight CS paired with a different food. Moreover, removal of the noncontingent foods led to complete recovery but more rapid extinction of responding to the same keylight relative to the different keylight. The implications of these results for understanding the mechanisms underlying depression and recovery of responding following degraded contingency and explicitly unpaired extinction procedures are discussed.