Training and testing determinants of short-term associative suppression of phototaxic behavior in Hermissenda

Hermissenda crassicornis shows both short- and long-term retention of conditioning following light-rotation pairings. Previous research has shown that prolonged training (50 trials per session, three consecutive daily sessions) produces a suppression of phototaxis lasting for days. This long-term retention reflects associative learning processes, with little or no contribution of nonassociative learning. In contrast, both associative learning and nonassociative behavioral modification contribute to short-term retention following a single session of five pairing trials. In this paper, we describe important associative and nonassociative determinants of short-term changes in phototaxis. In Experiment 1, animals received successive hourly tests for phototaxis in either a horizontal or a vertical orientation. Repeated testing resulted in decreased phototaxis which was especially pronounced for animals tested horizontally. Experiments 2-4 demonstrated that both repeated handling of animals and repeated periods of dark adaptation prior to each phototaxic test contributed to the development of phototaxic suppression with repeated testing. These nonassociative influences on phototaxis interacted with the gravitational orientation employed during behavioral testing, being most pronounced for testing in the horizontal orientation. An implication of these findings is that attempts to demonstrate short-term pairing-specific suppression will be most successful when nonassociative contributions are minimized (by testing animals vertically). Experiment 5 tested this prediction. We also tested the influence of training light intensity and the stimulus specificity of conditioned suppression. Animals received either five paired or five random presentations of light and rotation. Training light intensity was either moderate or bright. Following training, animals were tested for either suppression of phototaxis or suppression of negative geotaxis, using either a horizontal or a vertical testing orientation. Consistent with previous results, horizontally tested animals exhibited pronounced nonassociative suppression following training. The use of a bright training light also produced nonassociative suppression. However, when trained with a light of moderate intensity and tested vertically, Hermissenda showed associative suppression of phototaxis (significant paired--random difference) but not geotaxis (no paired--random difference). In a final experiment we observed that the longer the period of dark adaptation (prior to testing) the longer the phototaxic latency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Associatively reduced withdrawal from shadows in Hermissenda: a direct behavioral analog of photoreceptor responses to brief light steps

When the nudibranch Hermissenda crassicornis encounters a shadow in an otherwise uniformly illuminated field, it stops and turns back into the light within seconds. Associative conditioning, with paired light and rotation stimuli, produces learned modifications of phototaxis in illumination gradients. This same training procedure significantly reduced the ability of paired, but not random or naive control animals, to withdraw from shadows. In naive animals, after 13 min of dark adaptation, withdrawal from shadows was less apparent when animals encountered this stimulus the first time than after the second encounter. This difference in responsiveness to the first and second edge stimulus paralleled differences in type B photoreceptor impulse frequencies recorded during and after first and second steps of light. Earlier studies have shown that associative training of Hermissenda increases a long-lasting depolarization (LLD) which follows a light step. Our present findings suggest a functional relationship between the LLD of the type B photoreceptor and the behavioral response to light-dark differences. This supports the view that membrane changes which cause modifications of LLD magnitude store the learned association for later recall.     

Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis

Associative learning dependent on visual and vestibular sensory neurons and the underlying cellular mechanisms have been well characterized in Hermissenda but not yet in Lymnaea. Three days of conditioning with paired presentations of a light flash (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) in intact Lymnaea stagnalis results in a whole-body withdrawal response (WBWR) to the CS. In the current study, we examined the optimal stimulus conditions for associative learning, including developmental stage, number of stimuli, interstimulus interval, and intertrial interval. Animals with a shell length longer than 18 mm (sexually mature) acquired and retained the associative memory, while younger ones having a shell length shorter than 15 mm acquired but did not retain the memory to the following day. For mature animals, 10 paired presentations of the CS and UCS presented every 2 min were sufficient for the induction of a WBWR to the CS. Furthermore, animals conditioned with the UCS presented simultaneously with the last 2 s of the CS also exhibited a significant WBWR in response to the CS. Blind animals did not acquire the associative memory, suggesting that ocular photoreceptors, and not dermal photoreceptors, detected the CS. These results show that maturity was key to retention of associative learning.     

Contextually Based Conditional Discrimination of the Rabbit Eyeblink Response

Rabbits received conditional discrimination training using contextual stimuli to set the occasion for stimulus pairings during eyelid conditioning. Specifically, animals were exposed to either the presence or the absence of an oscillating chamber light throughout the intertrial interval (50 ± 10 s). For half the animals, this light signaled paired presentations of a discrete tone conditioned stimulus (CS) and air puff unconditioned stimulus (US) while darkness signaled presentations of only the tone CS. The remaining animals experienced the opposite contextual relationship to the conditioning stimuli. These trial types occurred pseudo-randomly across a session, with all transitions between contextual settings (i.e., light or dark) taking place immediately at the CS–US offset. Under these conditions, animals successfully utilized the contextual stimuli as conditional cues for differential responding to the shared CS. Moreover, both light and dark were equally effective as discriminative stimuli. A subset of animals received further training in which the contextual contingency was removed by restricting all conditioning to the CS-alone context. Without the contingency in place, subsequent CS presentations (paired and CS-alone) evoked equivalent conditioned responding across three sessions of training. Following the reinstatement of the contextual contingencies, discriminatory responding was immediately observed and returned to previous levels within three sessions. Finally, animals appeared to use the static representation of the conditional cue, rather than the phasic transition between cues, for discriminatory responding. These findings are discussed in terms of current neurobiological models of eyelid conditioning.     

Stimulus generalization of conditioned eyelid responses produced without cerebellar cortex: implications for plasticity in the cerebellar nuclei

In Pavlovian eyelid conditioning and adaptation of the vestibulo-ocular reflex, cerebellar cortex lesions fail to completely abolish previously acquired learning, indicating an additional site of plasticity in the deep cerebellar or vestibular nucleus. Three forms of plasticity are known to occur in the deep cerebellar nuclei: formation of new synapses, plasticity at existing synapses, and changes in intrinsic excitability. Only a cell-wide increase in excitability predicts that learning should generalize broadly from a training stimulus to other stimuli capable of supporting learning, whereas the alternatives predict that learning should be relatively specific to the training stimulus. Here we show that deep nucleus plasticity, as assessed by conditioned eyelid responses produced without input from the cerebellar cortex, is relatively specific to the training conditioned stimulus (CS). We trained rabbits to a tone or light CS with periorbital stimulation as the unconditioned stimulus (US), and pharmacologically disconnected the cerebellar cortex during a posttraining generalization test. The short-latency conditioned responses unmasked by this treatment showed strong decrement along the dimension of auditory frequency and did not generalize across stimulus modalities. These results cannot be explained solely by a cell-wide increase in the excitability of deep nucleus neurons, and imply that an input-specific mechanism in the deep cerebellar nucleus operates as well.     

Context-dependent olfactory learning in an insect

We studied the capability of the cricket Gryllus bimaculatus to select one of a pair of odors and to avoid the other in one context and to do the opposite in another context. One group of crickets was trained to associate one of a pair of odors (conditioned stimulus, CS1) with water reward (appetitive unconditioned stimulus, US+) and another odor (CS2) with saline solution (aversive US, US-) under illumination and to associate CS1 with US- and CS2 with US+ in the dark. Another group of crickets received training of the opposite stimulus arrangement. At 1 d after the training for 3 d, the former group significantly preferred CS1 over CS2 under illumination but preferred CS2 over CS1 in the dark, and the latter group exhibited the opposite odor preference. The results of control experiments showed that the background light condition had no significant effects on memory formation or retrieval unless it was explicitly associated with US during training. Thus, the visual context affected learning performance only when crickets were requested to use it to disambiguate the meaning of CSs and to predict USs.     

Pavlovian conditioning of Hermissenda: current cellular, molecular, and circuit perspectives

The less-complex central nervous system of many invertebrates make them attractive for not only the molecular analysis of the associative learning and memory, but also in determining how neural circuits are modified by learning to generate changes in behavior. The nudibranch mollusk Hermissenda crassicornis is a preparation that has contributed to an understanding of cellular and molecular mechanisms of Pavlovian conditioning. Identified neurons in the conditioned stimulus (CS) pathway have been studied in detail using biophysical, biochemical, and molecular techniques. These studies have resulted in the identification and characterization of specific membrane conductances contributing to enhanced excitability and synaptic facilitation in the CS pathway of conditioned animals. Second-messenger systems activated by the CS and US have been examined, and proteins that are regulated by one-trial and multi-trial Pavlovian conditioning have been identified in the CS pathway. The recent progress that has been made in the identification of the neural circuitry supporting the unconditioned response (UR) and conditioned response (CR) now provides for the opportunity to understand how Pavlovian conditioning is expressed in behavior.     

Reduction of two voltage-dependent K+ currents mediates retention of a learned association

A single identified neuron, the medial type B photoreceptor, was isolated by axotomy from the nervous systems of nudibranch molluscs (Hermissenda) which had been exposed to three different training experiences. Paired animals had been trained with repeated paired presentations of light and rotation and random animals with randomized light and rotation; naive animals had no training. A two-microelectrode voltage clamp of axotomized type B somata (separated from all synaptic interactions and impulse activity) was used to measure, with a blind procedure, three distinct ionic currents at least 24 h after the training experience. An early K+ current, IA, and a Ca2+-dependent K+ current, ICa2+-K+, but not a light-induced inward Na+ current, were significantly reduced for the paired as compared to the random and naive animals. The magnitude of ICa2+-K+ reduction was related (again measured blindly) to the degree of training-induced suppression of phototaxis (a measure of the learned behavior) for the paired animals. These data are consistent with previous observations indicating that changes of intrinsic type B membrane properties are an important means for encoding the acquisition and retention of Hermissenda associative learning.     

Classical Delay Eyeblink Conditioning in in 4- and 5-Month-Old Human Infants

Abstract-Simple delay classical eyeblink conditioning, using a tone conditioned stimulus (CS) and airpuff unconditioned stimulus (US), was studied in cross-sectional samples of 4- and 5-month-old healthy, full-term infants. Infants received two identical training sessions, 1 week apart. At both ages, infants experiencing paired tones and air-puffs demonstrated successful conditioning over two sessions, relative to control subjects who had unpaired training. Conditioning was not evident, however, during the first session. Two additional groups of 5-month-olds received varied experiences during Session 1, either unpaired presentations of the CS and US or no stimulus exposure, fol-lowed by paired conditioning during Session 2. Results from these groups suggest that the higher level of conditioning observed following two sessions of paired conditioning was not the result of familiarity with the testing environment or the stimuli involved but, rather, the result of retention of associative learning not expressed during the first conditioning session.     

Olfactory learning in the one-day old rat: reinforcing effects of isoproterenol

Within 24 h of their birth-induced norepinephrine surge, rat pups were tested for effects of a beta-receptor agonist, isoproterenol, on olfactory learning. Experiment 1 found no effect of isoproterenol on conditioning by pairing an odor (CS) with intraoral saccharin infusions. There was, however, unexpectedly strong responding in the unpaired control condition, which had the same contingency between the CS and isoproterenol as the paired condition. Experiment 2 found that pairings of odor and isoproterenol alone were sufficient for enhancing responding to the odor. Experiment 3 determined that isoproterenol had acted independently as a US for associative conditioning rather than facilitating nonassociative learning by mere exposure to the odor. These effects of isoproterenol as a US are consistent with the results of previous studies with older rats.     

Protein synthesis-dependent memory and neuronal enhancement in Hermissenda are contingent on parameters of training and retention

Following contiguous pairings of light and rotation, light alone elicits a conditioned contraction of Hermissenda's foot, indicative of an associative memory. After a 5-min retention interval, this conditioned response was evident following two or nine (but not one) conditioning trials but persisted for 90 min only after nine trials. In vivo incubation of animals in the protein synthesis inhibitor anisomycin (ANI; 1 microM) did not affect the conditioned response at the 5-min retention interval but significantly attenuated conditioned responding at the 90-min interval even following nine training trials. Deacetylanisomycin (DANI; 1 microM; an inactive form of anisomycin) had no effect on either 5- or 90-min retention. In a companion procedure, groups of isolated nervous systems were exposed to comparable light and rotation pairings, and the B photoreceptors (considered a site of storage for the associative memory) underwent electrophysiological analysis. An increase in neuronal excitability (indexed by depolarizing voltage responses to injected current) in the B photoreceptors paralleled the expression of conditioned responding in intact animals, that is, two training trials produced a short-term increase in excitability that dissipated within 45 min, whereas nine trials produced a persistent (at least 90-min) increase in excitability. In a fmal experiment, isolated nervous systems were exposed to nine training trials, and ANI or DANI was either present in the bathing medium before and during training or was introduced 5 min after training. Following training in ANI, a short-term (5- to 45-min) but not persistent (90-min) increase in excitability in the B photoreceptors was observed. ANI had no effect on either the short-term or persistent increase in excitability if the drug was applied 5 min after the last (ninth) training trial, and DANI had no effect on training-induced increases in excitability at any retention intervals. These results suggest that short-term retention in Hermissenda is protein synthesis independent but that new protein synthesis initiated during or shortly after the training event is necessary for even 90-min retention. Moreover, these results indicate that under some conditions, a critical threshold of training must be exceeded to initiate protein synthesis-dependent retention.     

Automated assessment of conditioning parameters for context and cued fear in mice

A behavioral technique often used to evaluate the cognitive performance of rats and mice is the fear conditioning paradigm. During conditioned fear experiments, freezing responses shown by rodents after exposure to environmental stimuli previously paired to an aversive experience provide a behavioral index of the animal's associative abilities. The present study examined the ability of a computer-controlled automated Freeze Monitor system for recording immobility behavior in mice. The sensitivity of the automated procedure to detect group differences caused by the application of various training protocols was also evaluated. Statistical analyses revealed significant positive correlations between immobility scores obtained with the automated apparatus and hand-scored data collected by a continuous or a time-sampling method. Behavioral patterns recorded by the computerized system were very similar to those obtained by the hand-scoring methods adopted. In particular, during context testing, exposure to environmental stimuli previously paired with a mild foot shock (unconditioned stimulus [US]) evoked increased immobility behavior in mice conditioned with the US compared with levels of immobility displayed by mice previously confined to the same contextual stimuli without receiving the US. Moreover, although during conditioned stimulus (CS) testing, mice previously exposed to the US displayed high levels of immobility when confined to environmental cues much different from those paired with the US (contextual fear generalization), both hand-scored and automated results revealed the effect of CS–US pairing (increased immobility) only in mice trained to associate the two stimuli (paired group) but not in mice exposed to both CS and US separated by a 40-sec time interval (unpaired group) or in mice receiving only the US (US group) during conditioning sessions. Overall, the results show associative conditioning measured in an automated apparatus and highlight the utility of obtaining both latency as well as beam interruption parameters.     

Context-dependent olfactory learning monitored by activities of salivary neurons in cockroaches

Context-dependent discrimination learning, a sophisticated form of nonelemental associative learning, has been found in many animals, including insects. The major purpose of this research is to establish a method for monitoring this form of nonelemental learning in rigidly restrained insects for investigation of underlying neural mechanisms. We report context-dependent olfactory learning (occasion-setting problem solving) of salivation, which can be monitored as activity changes of salivary neurons in immobilized cockroaches, Periplaneta americana. A group of cockroaches was trained to associate peppermint odor (conditioned stimulus, CS) with sucrose solution reward (unconditioned stimulus, US) while vanilla odor was presented alone without pairing with the US under a flickering light condition (1.0 Hz) and also trained to associate vanilla odor with sucrose reward while peppermint odor was presented alone under a steady light condition. After training, the responses of salivary neurons to the rewarded peppermint odor were significantly greater than those to the unrewarded vanilla odor under steady illumination and those to the rewarded vanilla odor was significantly greater than those to the unrewarded peppermint odor in the presence of flickering light. Similar context-dependent responses were observed in another group of cockroaches trained with the opposite stimulus arrangement. This study demonstrates context-dependent olfactory learning of salivation for the first time in any vertebrate and invertebrate species, which can be monitored by activity changes of salivary neurons in restrained cockroaches.     

Differential acquisition of conditioned suppression in rats with increased and decreased luminance levels as CS + s

Using a conditioned suppression procedure, it was demonstrated that suppression of responding was substantially enhanced when the CS signaling the occurrence of shock was an increment in luminance level (bright CS), relative to an equivalent decrement in luminance level serving as a signal for shock (dim CS). The results of a pseudoconditioning control procedure illustrated that the differential effectiveness of the bright and dim CSs was dependent upon explicit CS-US pairings. The bright and dim stimuli were found to produce equivalent rates of conditioning when they were used to signal the availability of reinforcing stimuli in an appetitive discrimination procedure. The interpretation considered to be most consistent with the data is associative in nature; rats may be brought into the experimental setting prepared to associate bright light with danger and contraprepared to associate dim light with danger.     

The comparator hypothesis of conditioned response generation: manifest conditioned excitation and inhibition as a function of relative excitatory strengths of CS and conditioning context at the time of testing

In the present research water-deprived rats were used in a conditioned lick suppression paradigm to test and further develop Rescorla's (1968) contingency theory, which posits that excitatory associations are formed when a conditioned stimulus (CS) signals an increase in unconditioned stimulus (US) likelihood and that inhibitory associations develop when the CS signals a decrease in US likelihood. In Experiment 1 we found that responding to a CS varied inversely with the associative status of the context in which the CS was trained and that this response was unaltered when testing occurred in a distinctively dissimilar context with a different conditioning history, provided associative summation with the test context was minimized. These results suggest that manifest excitatory and inhibitory conditioned responding is modulated by the associative value of the training context rather than that of the test context. In Experiment 2 it was demonstrated that postconditioning decreases in the associative value of the CS training context reduced the effective inhibitory value of the CS even when testing occurred outside of the training context. Moreover, this contextual deflation effect was specific to the CS training context as opposed to any other excitatory context. Collectively, these studies support the comparator hypothesis, which states that conditioned responding is determined by a comparison of the associative strengths of the CS and its training context that occurs at the time of testing rather than at the time of conditioning. This implies that all associations are excitatory and that responding indicative of conditioned inhibition reflects a CS-US association that is below (or near) the associative strength of its comparator stimulus. It is suggested that response rules which go beyond a monotonic relation between associative value and response strength can partially relieve learning theories of their explanatory burdens, thereby allowing for simpler models of acquisition.     

Feeding behavior of Aplysia: a model system for comparing cellular mechanisms of classical and operant conditioning

Feeding behavior of Aplysia provides an excellent model system for analyzing and comparing mechanisms underlying appetitive classical conditioning and reward operant conditioning. Behavioral protocols have been developed for both forms of associative learning, both of which increase the occurrence of biting following training. Because the neural circuitry that mediates the behavior is well characterized and amenable to detailed cellular analyses, substantial progress has been made toward a comparative analysis of the cellular mechanisms underlying these two forms of associative learning. Both forms of associative learning use the same reinforcement pathway (the esophageal nerve, En) and the same reinforcement transmitter (dopamine, DA). In addition, at least one cellular locus of plasticity (cell B51) is modified by both forms of associative learning. However, the two forms of associative learning have opposite effects on B51. Classical conditioning decreases the excitability of B51, whereas operant conditioning increases the excitability of B51. Thus, the approach of using two forms of associative learning to modify a single behavior, which is mediated by an analytically tractable neural circuit, is revealing similarities and differences in the mechanisms that underlie classical and operant conditioning.     

Amount of training and cue-evoked taste-reactivity responding in reinforcer devaluation

In two experiments, rats received minimal (16) pairings of one auditory conditioned stimulus (CS) cue with a sucrose reinforcer, and extensive (112) pairings of another auditory CS with that reinforcer. After sucrose was devalued by pairing it with lithium chloride in some rats (Devalue groups) but not others (Maintain groups), taste reactivity (TR) and other responses to unflavored water were assessed in the presence of the auditory CSs alone. The minimally trained CS controlled substantially more evaluative TR responses than the extensively trained CS. Those TR responses were hedonic (positive) in the Maintain groups, but aversive (negative) in the Devalue groups. By contrast, food cup entry and other responses thought not to reflect evaluative taste processing were controlled more by the extensively trained cue. These responses were reduced by sucrose devaluation comparably, regardless of the amount of training. The results suggest rapid changes in the content of learning as conditioning proceeds. Early in training, CSs may be capable of activating preevaluative processing of an absent food reinforcer that includes information about its palatability, but that capability is lost as training proceeds.     

Active avoidance conditioning in zebrafish (Danio rerio)

The zebrafish represents a potentially useful organism for studying genes involved in learning and memory function in vertebrates, because a number of genetic techniques in zebrafish have been developed to produce a wide variety of genetic mutants. While zebrafish mutants are being developed, behavioral studies on learning and memory function in zebrafish are in urgent need. The present study investigated active avoidance conditioning in normal zebrafish. Zebrafish were trained to swim from a lighted (CS) compartment to a dark compartment to avoid an electrical body shock (US) in a shuttle-box that consisted of a water-filled tank separated by an opaque barrier into two equal compartments. By varying the number of trials per training session and the duration of the intertrial interval, Experiments 1 and 2 showed that, with the CS, US, and intertrial interval being 12s, zebrafish learned avoidance responses within a training session consisting of 30 trials and retained the avoidance responses. Experiment 3 showed that zebrafish learned avoidance responses following the association between the CS of light and the US of shock in the avoidance conditioning paradigm. Using the avoidance conditioning paradigm, Experiment 4 investigated the amnestic effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor L-NAME in zebrafish. Experiment 4 showed that post-training injection of L-NAME significantly impaired retention of avoidance responses while MK-801 did not, confirming previous results with other vertebrates. The results of the present study suggest the similar involvements of neurochemicals in learning and memory among vertebrates. Thus, future studies with zebrafish mutants may identify genes involved in learning and memory in vertebrates.     

The strength of the orienting response during blocking

In two experiments rats initially received appetitive Pavlovian conditioning with an auditory conditioned stimulus (CS). Subsequently this stimulus was presented in compound with a novel light and paired with the same appetitive reinforcer. In keeping with the outcome of many such experiments on blocking, there was very little evidence of appetitive conditioning during subsequent independent presentations of the light. Of main concern in the present experiments, however, was the influence of this training on the strength of the orienting response directed towards the light. When the light was first presented it elicited a strong orienting response. The strength of this response declined rapidly when the light was presented in compound with the previously trained auditory CS but more slowly when it was paired with the reinforcer either by itself or in conjunction with an initially neutral auditory stimulus. It is suggested that the extent to which the events following the light are accurately predicted determines the strength of orientation towards this stimulus.     

Reevaluating evaluative conditioning: a nonassociative explanation of conditioning effects in the visual evaluative conditioning paradigm.

In 2 studies, the authors investigated whether evaluative conditioning (EC) is an associative phenomenon. Experiment 1 compared a standard EC paradigm with nonpaired and no-treatment control conditions. EC effects were obtained only when the conditioned stimulus (CS) and unconditioned stimulus (UCS) were rated as perceptually similar. However, similar EC effects were obtained in both control groups. An earlier failure to obtain EC effects was reanalyzed in Experiment 2. Conditioning-like effects were found when comparing a CS with the most perceptually similar UCSs used in the procedure but not when analyzing a CS rating with respect to the UCS with which it was paired during conditioning. The implications are that EC effects found in many studies are not due to associative learning and that the special characteristics of EC (conditioning without awareness and resistance to extinction) are probably nonassociative artifacts of the EC paradigm.