Discriminative behavior and Pavlovian conditioning in the mollusc Pleurobranchaea

The buccal motor system in the sea slug Pleurobranchaea californica is multifunctional; similar sets of neurons and muscles generate different behaviors through similar electrophysiological motor patterns. Such multifunctional systems compromise the traditional practice of identifying a motor pattern and then using that pattern to indicate the behavior in reduced preparations. We address this issue in a series of experiments leading to the comparison of differential Pavlovian conditioning in whole animals with the conditioned behavior of the same animals during electrophysiological recording. Because differential conditioning requires two conditioned stimuli (CSs), we show here that each of two CSs activated the conditioned response from animals after they received the stimulus (CS+) paired with an unconditioned stimulus (UCS). Conditioning sessions consisted of 5 training trials with a 2-h intertrial interval. In one study, experimental animals received a 60-s CS+, derived from beer (Sbr), paired with a 50-s electrical shock UCS whose onset occurred 10 s after the CS+ onset; control animals received the Sbr and UCS explicitly unpaired. In a second study, animals received similar procedures as in the first but with a CS+ consisting of squid homogenate (Ssq). Tests with both CSs showed that animals did not discriminate between Sbr and Ssq before beginning conditioning, but did so afterward. Experimental animals exhibited robust food aversion (withdrawal and suppressed feeding) to the CS+, but retained strong appetitive responses to the CS they did not receive in training; response thresholds to the CS+ changed as much as 1000-fold by comparison to the preconditioning values. Control animals exhibited similar though significantly smaller behavioral changes as the experimental animals. Both stimuli evoked associatively learned responses, but Sbr produced greater experimental-control differences than Ssq did. Two accompanying papers show the results of using both CSs in differential conditioning, and describe the behavioral/electrophysiological comparisons.     

Comparison of differential Pavlovian conditioning in whole animals and physiological preparations of Pleurobranchaea: implications of motor pattern variability

The present study compares differential Pavlovian conditioning in whole animals with the behavior of the same animals during electrophysiological recording. Untrained specimens of the sea slug Pleurobranchaea did not discriminate between two appetitive stimuli, one derived from an extract of beer (Sbr) and the other from a homogenate of squid muscle (Ssq). When animals received Sbr as the CS+ and Ssq as the CS- in a single day of five-trial, differential Pavlovian conditioning they learned to avoid selectively the Sbr but continued to exhibit appetitive responses to Ssq. Quantitative measures show that there was over a 1000-fold increase in the thresholds of the proboscis extension and bite-strike responses, many animals ceased all feeding behavior, and exhibited withdrawal responses to Sbr. We examined the behavior of the same trained animals immediately before preparing them for physiological recording and during the recording session. There was a close one-to-one relationship between these behavioral observations, showing that the qualitative and quantitative features of whole-animal Pavlovian conditioning persist into the physiological preparations. Unexpectedly, motor patterns from untrained preparations showed considerable variability both within the same preparation at different times and between preparations; conditioning appeared to increase such variability. Thus, it was not possible to state unequivocally the behavior of the animal by examining the electromyogram recording alone. Many of the trained preparations not only exhibited suppressed feeding behavior and withdrawal responses to Sbr, but, as a consequence of the multifunctional nature of the Pleurobranchaea buccal-oral system, also regurgitated previously ingested Ssq or squid meat when they were stimulated with Sbr. We discuss the findings with respect to self-organizing mechanisms that may establish motor patterns in multifunctional systems, and suggest that such mechanisms may lead to the generation of behaviors that are not specifically encoded by the conditioned cellular changes.     

Innate and enhanced predator recognition in hatchery-reared chinook salmon

We used a laboratory behaviour assay to investigate how innate predator recognition, handling stress, retention time, and number of conditioning events might affect chemically mediated anti-predator conditioning for hatchery-reared chinook salmon, Oncorhynchus tshawytscha. Juvenile chinook salmon with no prior exposure to predatory stimuli exhibited innate fright responses to northern pikeminnow, Ptychocheilis oregonensis, odour, regardless of whether the salmon came from a population that exists in sympatry or allopatry with northern pikeminnows. Juvenile chinook salmon exhibited enhanced predator recognition following a single conditioning event with conspecific extract and northern pikeminnow odour. Handling similar to what hatchery salmon might experience prior to release did not substantially reduce the conditioned response. When we conditioned juvenile chinook salmon in hatchery rearing vessels, fish from tanks treated once exhibited a conditioned response to northern pikeminnow odour in aquaria, but only for one behaviour (feeding response), and fish treated twice did not respond. The results suggest that enhanced recognition of predator stimuli occurs quickly, but may be to some extent context-specific, which may limit conditioned fright responses after release into the natural environment.     

Exposures to conditioned flavours with different hedonic values induce contrasted behavioural and brain responses in pigs

This study investigated the behavioural and brain responses towards conditioned flavours with different hedonic values in juvenile pigs. Twelve 30-kg pigs were given four three-day conditioning sessions: they received three different flavoured meals paired with intraduodenal (i.d.) infusions of 15% glucose (F(Glu)), lithium chloride (F(LiCl)), or saline (control treatment, F(NaCl)). One and five weeks later, the animals were subjected to three two-choice feeding tests without reinforcement to check the acquisition of a conditioned flavour preference or aversion. In between, the anaesthetised pigs were subjected to three (18)FDG PET brain imaging coupled with an olfactogustatory stimulation with the conditioned flavours. During conditioning, the pigs spent more time lying inactive, and investigated their environment less after the F(LiCl) than the F(NaCl) or F(Glu) meals. During the two-choice tests performed one and five weeks later, the F(NaCl) and F(Glu) foods were significantly preferred over the F(LICl) food even in the absence of i.d. infusions. Surprisingly, the F(NaCl) food was also preferred over the F(Glu) food during the first test only, suggesting that, while LiCl i.d. infusions led to a strong flavour aversion, glucose infusions failed to induce flavour preference. As for brain imaging results, exposure to aversive or less preferred flavours triggered global deactivation of the prefrontal cortex, specific activation of the posterior cingulate cortex, as well as asymmetric brain responses in the basal nuclei and the temporal gyrus. In conclusion, postingestive visceral stimuli can modulate the flavour/food hedonism and further feeding choices. Exposure to flavours with different hedonic values induced metabolism differences in neural circuits known to be involved in humans in the characterization of food palatability, feeding motivation, reward expectation, and more generally in the regulation of food intake.     

Concomitant Pavlovian conditioning of heart rate and leg flexion responses in the rat

Pavlovian conditioning was studied in male Fischer 344 rats using tones as the conditioned stimulus (CS) and footshock as the unconditioned stimulus (UCS). Different groups of animals received (a) contiguous CS-UCS pairings with a 0.5 sec CS, (b) contiguous CS-UCS pairings with a 4.0 sec CS, or (c) a random sequence of noncontiguous tones and shocks using either a 0.5 sec or a 4.0 sec CS. Heart rate (HR) and leg flexion (LF) responses were recorded. Leg flexion conditioning occurred only in the 0.5 sec contiguous group. Decelerative HR CRs occurred only in the 4.0 sec contiguous group. Accelerative HR changes occurred in the other two groups but were significantly greater in the 0.5 sec contiguous group. These results are similar to but not identical to those obtained during eyeblink or nictitating membrane conditioning in rabbits, and suggest that the topography of the Pavlovian HR CR is dependent on the simultaneous occurrence of other classically conditioned responses.     

The characteristics of the electrovomeronasogram: its loss following vomeronasal axotomy in the garter snake

Electrovomeronasogram (EVG) recordings were made from adult garter snakes, Thamnophis sirtalis. Stimulation of vomeronasal epithelium with a stimulus prepared from prey, earthworm electric shock secretion (ESS), evoked EVG response in a dose-dependent manner. The magnitude of the EVG response to ESS was remarkably larger than n-amyl acetate and glutamate, which elicited insignificant responses, supporting the idea that the vomeronasal system is differentially sensitive to liquid delivery of biologically significant chemical stimuli. Fourteen days following vomeronasal axotomy, the magnitudes of the EVG responses of animals which received bilateral axotomy without cauterization or with cauterization was -0.19+/-0.07 mV or -0.05+/-0.02 mV respectively, compared with the normal EVG response of -0.41+/-0.10 mV. The epithelia of animals which received bilateral axotomy without cauterization exhibited remarkable degeneration of the bipolar neurons. Maximal depletion of bipolar neurons occurred in the epithelia denervated with cauterization, though the difference between cell densities in vomeronasal neuron layers in these epithelia was not statistically significant. The present results clearly indicate that the fewer neurons the epithelium contains, the smaller EVG response it generates, suggesting that the receptor neurons are the primary origin of EVG responses.      

Klassische Konditionierung agonistischen Verhaltens beim Syrischen Goldhamster (Mesocricetus auratus Waterhouse)

Nine different reactions of unconditioned agonistic behavior of golden hamsters (Mesocricetus auratus Waterhouse) which could be elicited by an air blow (UCS) were classically conditioned at a sound of a xylophone (tone g), a previously neutral stimulus. Tone g (CS) was paired with UCS for a different number of trials/day. In all animals exposed to over 20 pairings/day components of the agonistic behavior were brought under the control of Cs, but it was not possible to predict which one of the agonistic reactions would appear. Most of the animals generalized and also showed conditioned reactions (CR) when presenting tone c' instead of tone g. When utilizing a differential conditioning procedure the animals learned to discriminate between the different tones. Extinction of CR at tone g was reached after an individually different number of trials.     

Worrying affects associative fear learning: a startle fear conditioning study

A valuable experimental model for the pathogenesis of anxiety disorders is that they originate from a learned association between an intrinsically non-aversive event (Conditioned Stimulus, CS) and an anticipated disaster (Unconditioned Stimulus, UCS). Most anxiety disorders, however, do not evolve from a traumatic experience. Insights from neuroscience show that memory can be modified post-learning, which may elucidate how pathological fear can develop after relatively mild aversive events. Worrying--a process frequently observed in anxiety disorders--is a potential candidate to strengthen the formation of fear memory after learning. Here we tested in a discriminative fear conditioning procedure whether worry strengthens associative fear memory. Participants were randomly assigned to either a Worry (n = 23) or Control condition (n = 25). After fear acquisition, the participants in the Worry condition processed six worrisome questions regarding the personal aversive consequences of an electric stimulus (UCS), whereas the Control condition received difficult but neutral questions. Subsequently, extinction, reinstatement and re-extinction of fear were tested. Conditioned responding was measured by fear-potentiated startle (FPS), skin conductance (SCR) and UCS expectancy ratings. Our main results demonstrate that worrying resulted in increased fear responses (FPS) to both the feared stimulus (CS(+)) and the originally safe stimulus (CS(-)), whereas FPS remained unchanged in the Control condition. In addition, worrying impaired both extinction and re-extinction learning of UCS expectancy. The implication of our findings is that they show how worry may contribute to the development of anxiety disorders by affecting associative fear learning.     

Tailflick escape behavior in larval and juvenile lobsters (Homarus americanus) and crayfish (Cherax destructor)

We examined the escape behavior of larvae and postlarvae of the American lobster (Homarus americanus) and of adult immature (stage ADI) crayfish (Cherax destructor). Responses to standardized water jet stimuli delivered through a pipette were observed and analyzed. Lobster larvae did not respond to stimuli within 60 ms, indicating that they do not have functional giant fibers. The first movement by lobster larvae in response to water jet stimuli was a hyperextension of the abdomen. Larval escape responses also showed very little habituation. Postlarval lobsters and ADI crayfish showed the same range of responses as adult animals. Displacement efficiency of tailflicks exhibited by the different animals and stages was examined and related to the morphology of the animals. A separate behavior from tailflicking by larval lobsters in response to water jet stimuli was also observed. Here, the abdomen was hyperextended and the thoracic appendages were promoted. We termed this behavior a "starburst" response. The features of the tailflicking behavior suggest that it evolved to make the larvae difficult prey to handle for small, slower moving predators, and possibly to allow them to ride the bow waves of faster moving predators.     

Pavlovian conditioning: pigeon nictitating membrane

A new Pavlovian conditioning preparation was developed using the nictitating membrane of the restrained pigeon. Either visual or auditory stimuli served as conditioned stimuli (CSs) with an unconditioned stimulus (US) of a puff of air to the cornea. Movement of the nictitating membrane constituted the conditioned and unconditioned responses (CR and UR). Conditioning was studied with the Kamin blocking procedure. In agreement with findings from other conditioning preparations, responding to the redundant stimulus was attenuated relative to a stimulus that received the same number of CS-US pairings in a compound-conditioning procedure. Although response attenuation occurred, substantial individual variation was observed within the blocking procedure, a finding with some precedent in the experimental literature. Theoretical analysis and neural-network simulations indicate that inter-subject variation in response attenuation may result from differences in the extent to which contextual stimuli contribute to the functional CS.     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

Reaction time task as unconditional stimulus. Comparing aversive and nonaversive unconditional stimuli

Nonaversive unconditional stimuli (USs) are seldom used in human classic conditioning of autonomic responses. One major objection to their use is that they produce deficits in electrodermal (ED) second- and third-interval response conditioning. However, a nonaversive reaction time (RT) task that includes feedback of success has been shown to be an effective US while avoiding this disadvantage (Lipp and Vaitl 1988). The present study compared this new RT task (RT-new) with a traditional RT task (RT-old) and with a standard aversive US (shock) in differential classic conditioning of ED, heart rate (HR), and digital pulse volume (DPV) responses. Eight-second-delay differential conditioning was applied in three groups of 12 subjects each. Simple geometric features (square, cross) displayed on a television screen served as conditional stimuli (CS+ and CS-). In acquisition, there were no statistically significant differences among the groups; differential conditioning did occur in HR, first- and second-interval ED responses, and first-interval DPV responses. Separate analyses within each group, however, revealed that there was no second-interval ED conditioning in the RT-old group. During extinction, neither DPV nor second-interval ED conditioning could be obtained, whereas HR and first-interval ED conditioning occurred in each group. In third-interval omission ED responses, RT-old and shock groups exhibited extinction, while response differentiation was maintained in the RT-new group throughout extinction. The RT task including feedback proved to be as reliable a US as a standard aversive US, whereas application of a traditional RT task again yielded some weaknesses in second-interval ED conditioning.     

An Analysis of Variability in the Feeding Motor Program of the Honey Bee; the Role of Learning in Releasing a Modal Action Pattern

Sequences of behavior are highly predictable (stereotyped) during some segments and less predictable during transitions between those segments. Statistical characterization of behavior must involve observation of the behavior under different stimulus conditions, which includes how stimuli associated with behavioral releasers come to trigger behavior during learning. For example, the feeding motor program (FMP) of the honey bee Apis mellifera (Hymenoptera: Apidae) during proboscis extension can be divided into three response phases (Rehder 1987). We conditioned honey bees in an olfactory conditioning paradigm with one or several rewarded trials during which an odorant was paired with the sugar-water unconditioned stimulus (US); the latter elicits proboscis extension and feeding in properly motivated bees. By recording electromyogram activity from one of the muscles that move the proboscis during feeding, we quantified the bees' responses during an unrewarded test with either the conditioned odorant, a different (novel) odorant, or the sugar-water US. Various parameters of the response phases of the FMP varied in a consistent manner across these experimental treatments, with certain stimuli eliciting stronger, more consistent responses. The different response phases followed one another in time with some variability and statistical uncertainty. For example, the length of an individual licking movement with the glossa was relatively invariant, and may indicate that this parameter can be used to differentiate the FMP into more basic, independent units. Our work shows how learned information may release action patterns in ways slightly different from traditional sign-stimuli releasers.     

In vitro classical conditioning of a gill withdrawal reflex in Aplysia: neural correlates and possible neural mechanisms

An in vitro preparation consisting of the siphon, mantle, gill, and abdominal ganglion undergoes classical conditioning when a weak tactile stimulus (CS) applied to the siphon is paired with a strong tactile stimulus to the gill (UCS). When the stimuli are paired, the CS comes to evoke a gill withdrawal reflex (GWR) which increases in amplitude with training. Only when the stimuli are paired in a classical conditioning paradigm does the CS come to evoke a GWR. With classical conditioning training there is an alteration in the synaptic efficacy between central sensory neurons and central gill motor neurons. Moreover, these changes can be observed in sensory neurons not activated by the CS. The changes observed, as evidence by the number of action potentials evoked in the gill motor neuron do not completely parallel the observed behavioral changes. It is suggested that in addition to changes in the synaptic efficacy at the sensory-motor neuron synapse, other changes in neuronal activity occur at other loci which lead to the observed behavioral changes.     

Water jet: a simple method for classical conditioning in fish

Classical conditioning in animals is a learning procedure involving a biologically relevant stimulus paired with a previously neutral stimulus. In fish, light and sound are frequently used as previously neutral stimuli for conditioning tests. However, in laboratory experiments with replicates, such stimuli may influence the responses of fish in nearby aquariums. Herein, we developed a simple applicable methodology for classical conditioning in fish that prevents this type of influence. We isolated fish in individual aquariums and introduced a water jet that caused localized water movement, followed by the introduction of a food pellet. These procedures were repeated for each fish for 20 days. After 14 days, all fish were conditioned. Moreover, in subsequent probe trials (memory retention tests) conducted within 32 days after conditioning procedures, fish responded accordingly. These findings corroborate the applicability and usefulness of the method tested herein especially under lab conditions. Therefore, we suggest that a simple water jet is a useful and reliable tool for fish conditioning in future studies.     

Varying temporal placement during CS of an added stimulus correlated with non-delivery of UCS.

This experiment extends Pavlov's method of contrasts for training a stimulus discrimination to the case of the cardiac conditional response in the rhesus monkey. It explores the parameter of temporal placement of an additional stimulus ("CS2") within a 10-sec CS (or "CS1"), with the appearance of the former stimulus on any trial signalling the absence of UCS (electric shock) on that trial. This experimental paradigm is a parallel to that of the "intruded stimulus" studies in operant conditioning. In both cases, several ways of describing the function of the added stimulus are possible, but all seem reducible to the same operational terms. Data were taken in the present study with respect to the form and latency of the cardiac rate changes produced by intrusion of CS2 (light), across a range of placements varying from simultaneity with CS1 (a different light) onset to two sec before UCS would have been delivered. The control of CS2 over the cardiac rate CR was occasionally exhibited with a latency as short as three beats after stimulus onset. The order of CS2 temporal placements to which a subject was exposed was a factor in determining the form of the conditioned cardiac rate response to CS1.     

CB(1) Receptor Autoradiographic Characterization of the Individual Differences in Approach and Avoidance Motivation

Typically, approach behaviour is displayed in the context of moving towards a desired goal, while avoidance behaviour is displayed in the context of moving away from threatening or novel stimuli. In the current research, we detected three sub-populations of C57BL/6J mice that spontaneously responded with avoiding, balancing or approaching behaviours in the presence of the same conflicting stimuli. While the balancing animals reacted with balanced responses between approach and avoidance, the avoiding or approaching animals exhibited inhibitory or advance responses towards one of the conflicting inputs, respectively. Individual differences in approach and avoidance motivation might be modulated by the normal variance in the level of functioning of different systems, such as endocannabinoid system (ECS). The present research was aimed at analysing the ECS involvement on approach and avoidance behavioural processes. To this aim, in the three selected sub-populations of mice that exhibited avoiding or balancing or approaching responses in an approach/avoidance Y-maze we analysed density and functionality of CB(1) receptors as well as enzyme fatty acid amide hydrolase activity in different brain regions, including the networks functionally responsible for emotional and motivational control. The main finding of the present study demonstrates that in both approaching and avoiding animals higher CB(1) receptor density in the amygdaloidal centro-medial nuclei and in the hypothalamic ventro-medial nucleus was found when compared with the CB(1) receptor density exhibited by the balancing animals. The characterization of the individual differences to respond in a motivationally based manner is relevant to clarify how the individual differences in ECS activity are associated with differences in motivational and affective functioning.     

Conditioning and sexual behavior: a review

Sexual behavior is directed by a sophisticated interplay between steroid hormone actions in the brain that give rise to sexual arousability and experience with sexual reward that gives rise to expectations of competent sexual activity, sexual desire, arousal, and performance. Sexual experience allows animals to form instrumental associations between internal or external stimuli and behaviors that lead to different sexual rewards. Furthermore, Pavlovian associations between internal and external stimuli allow animals to predict sexual outcomes. These two types of learning build upon instinctual mechanisms to create distinctive, and seemingly "automated," patterns of sexual response. This article reviews the literature on conditioning and sexual behavior with a particular emphasis on incentive sequences of sexual behavior that move animals from distal to proximal with regard to sexual stimuli during appetitive phases of behavior and ultimately result in copulatory interaction and mating during consummatory phases of behavior. Accordingly, the role of learning in sexual excitement, in behaviors that bring about the opportunity to mate, in courtship and solicitation displays, in sexual arousal and copulatory behaviors, in sexual partner preferences, and the short- and long-term influence of copulatory experience on sexual and reproductive function is examined. Although hormone actions set the stage for sexual activity by generating the ability of animals to become sexually excited and aroused, it is each animal's unique experience with sexual behavior and sexual reward that molds the strength of responses made toward sexual incentives.     

Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees

To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals.     

Oscillators entrained by food and the emergence of anticipatory timing behaviors

Circadian rhythms are adjusted to the external environment by the light-dark cycle via the suprachiasmatic nucleus, and to the internal environment of the body by multiple cues that derive from feeding/fasting. These cues determine the timing of sleep/wake cycles and all the activities associated with these states. We suggest that numerous sources of temporal information, including hormonal cues such as corticoids, insulin, and ghrelin, as well as conditioned learned responses determined by the temporal relationships between photic and feeding/fasting signals, can determine the timing of regularly recurring circadian responses. We further propose that these temporal signals can act additively to modulate the pattern of daily activity. Based on such reasoning, we describe the rationale and methodology for separating the influences of these diverse sources of temporal information. The evidence indicates that there are individual differences in sensitivity to internal and external signals that vary over circadian time, time since the previous meal, time until the next meal, or with duration of food deprivation. All of these cues are integrated in sites and circuits modulating physiology and behavior. Individuals detect changes in internal and external signals, interpret those changes as "hunger," and adjust their physiological responses and activity levels accordingly.