Multiunit changes in hippocampus and medial geniculate body in free-behaving rats during acquisition and retention of a conditioned response to a tone

Multiunit activity (MUA) was recorded chronically in the hippocampus (CA3) and the medial geniculate body (mMG) during habituation to a tone followed by conditioning (tone paired with footshock) or pseudoconditioning (tone/footshock unpaired) in rats previously trained in a lever-pressing for food task (VI 60). In the conditioned group pairing tones with footshocks rapidly induced an increase in the initial CS-evoked response in the mMG, followed by the emergence of a hippocampal response and a marked conditioned suppression of lever-pressing to the tone. In contrast, in the pseudoconditioned group, the stimulus induced only transient cellular changes in the hippocampus and in the mMG, while no behavioral suppression to the tone could be seen. Moreover, presentations of the CS 45 days later induced multiunit and behavioral responses in both structures, only in the conditioned group. These results are used for discussion of the role of learning-induced changes in the sensory structure (mMG) as compared with changes in an associative structure (hippocampus), during acquisition and retention of a conditioned response.     

Differential preparatory cardiovascular responses to aversive and appetitive behavioral conditioning

Dogs were required to press a response panel either to avoid shock or obtain food. During a one-hour interval immediatelypreceding performance on the avoidance task, blood pressure increased while heart rate decreased. In contrast, both blood pressure and heart rate increased during the one-hour interval immediatelypreceding performance on the food task. During both the shock-avoidance and food performancesper se, however, elevations in heart rate and blood pressure were observed, though the avoidance task produced less consistent heart rate changes. These effects were observed (1) with different individual dogs on each of the two training procedures, and (2) with the same dogs exposed successively to both aversive and appetitive behavioral conditioning.     

In vitro analog of classical conditioning of feeding behavior in aplysia

The feeding behavior of Aplysia californica can be classically conditioned using tactile stimulation of the lips as a conditioned stimulus (CS) and food as an unconditioned stimulus (US). Moreover, several neural correlates of classical conditioning have been identified. The present study extended previous work by developing an in vitro analog of classical conditioning and by investigating pairing-specific changes in neuronal and synaptic properties. The preparation consisted of the isolated cerebral and buccal ganglia. Electrical stimulation of a lip nerve (AT4) and a branch of the esophageal nerve (En2) served as the CS and US, respectively. Three protocols were used: paired, unpaired, and US alone. Only the paired protocol produced a significant increase in CS-evoked fictive feeding. At the cellular level, classical conditioning enhanced the magnitude of the CS-evoked synaptic input to pattern-initiating neuron B31/32. In addition, paired training enhanced both the magnitude of the CS-evoked synaptic input and the CS-evoked spike activity in command-like neuron CBI-2. The in vitro analog of classical conditioning reproduced all of the cellular changes that previously were identified following behavioral conditioning and has led to the identification of several new learning-related neural changes. In addition, the pairing-specific enhancement of the CS response in CBI-2 indicates that some aspects of associative plasticity may occur at the level of the cerebral sensory neurons.     

Fear develops to the conditioned stimulus and to the context during classical eyeblink conditioning in rats

In classical eyeblink conditioning, non-specific emotional responses to the aversive shock unconditioned stimulus (US), which are presumed to coincide with the development of fear, occur early in conditioning and precede the emergence of eyeblink responses. This twoprocess learning model was examined by concurrently measuring fear and eyeblink conditioning in the freely moving rat. Freezing served as an index of fear in animals and was measured during the inter-trial intervals in the training context and during a tone conditioned stimulus (CS) presented in a novel context. Animals that received CS-US pairings exhibited elevated levels of fear to the context and CS early in training that decreased over sessions, while eyeblink conditioned responses (CRs) developed gradually during acquisition and decreased during extinction. Random CS-US presentations produced a similar pattern of fear responses to the context and CS as paired presentations despite low eyeblink CR percentages, indicating that fear responding was decreased independent of high levels of learned eyeblink responding The results of paired training were consistent with two-process models of conditioning that postulate that early emotional responding facilitates subsequent motor learning, but measures from random control animals demonstrate that partial CS-US contingencies produce decrements in fear despite low levels of eyeblink CRs. These findings suggest, a relationship between CS-US contingency and fear levels during eyeblink conditioning, and may serve to clarify further the role that fear conditioning plays in this simple paradigm.     

Fear Develops to the Conditioned Stimulus and to the Context during Classical Eyeblink Conditioning in Rats

In classical eyeblink conditioning, non-specific emotional responses to the aversive shock unconditioned stimulus (US), which are presumed to coincide with the development of fear, occur early in conditioning and precede the emergence of eyeblink responses. This two-process learning model was examined by concurrently measuring fear and eyeblink conditioning in the freely moving rat. Freezing served as an index of fear in animals and was measured during the inter-trial intervals in the training context and during a tone conditioned stimulus (CS) presented in a novel context. Animals that received CS-US pairings exhibited elevated levels of fear to the context and CS early in training that decreased over sessions, while eyeblink conditioned responses (CRs) developed gradually during acquisition and decreased during extinction. Random CS-US presentations produced a similar pattern of fear responses to the context and CS as paired presentations despite low eyeblink CR percentages, indicating that fear responding was decreased independent of high levels of learned eyeblink responding. The results of paired training were consistent with two-process models of conditioning that postulate that early emotional responding facilitates subsequent motor learning, but measures from random control animals demonstrate that partial CS-US contingencies produce decrements in fear despite low levels of eyeblink CRs. These findings suggest a relationship between CS-US contingency and fear levels during eyeblink conditioning, and may serve to clarify further the role that fear conditioning plays in this simple paradigm.     

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.     

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.     

Classical conditioning of the nictitating membrane response of the lemon shark (Negaprion brevirostris)

An experimental group of lemon sharks received 100 daily presentations of light flash as the conditioned stimulus (CS) and electric shock as the unconditioned stimulus (US) in a classical conditioning situation. The conditioned responses (CRs) and unconditioned responses (URs) under observation consisted of extensions of the nictitating membrane. Separate control groups received either (a) no CS or US, (b) CS-alone, or (c) completely random presentations of CS and US. Few CRs occurred in the experimental group at the outset of conditioning, but the percentage of CRs during the second half of the first acquisition session exceeded 95%. Conditioning stabilized above 95% CRs during Acquisition Sessions 3 through 7. These responses could not be attributed to pseudoconditioning, sensitization, or other nonassociative factors. When the experimental group was subsequently given six CS-alone sessions, the course of extinction was gradual. Most results seemed similar to those previously obtained during classical conditioning of the nictitating membrane in rabbits.     

The influence of positive and negative reinforcement on selective attention in the rat

In Experiments 1 and 2 rats were trained under two multiple schedules of reinforcement. In one, bar pressing during a tone-light compound stimulus was reinforced under a variable-interval food reinforcement schedule. In the other multiple schedule, bar pressing avoided grid shock on a free-operant schedule. In both multiple schedules, a discrimination was maintained by an extinction schedule that was operative during the absence of the tone-light compound. In Experiments 1 and 2 the intensity of the tone-light compound was manipulated over three levels. Subsequent extinction tests revealed that light was attended to, almost exclusively of the tone, when food reinforcement had maintained bar pressing. On the other hand, the tone gained considerable attentional control under the shock avoidance schedule. This stimulus-reinforcer interaction was maintained for all three levels of the compound intensity. In Experiment 3 it was investigated whether this interaction was associative by presenting shock during the absence of the tone-light compound when food reinforcement maintained responding, and food during the absence of the compound when shock avoidance maintained responding. Since both food and shock were presented during a single session for both schedules, nonassociative effects of the reinforcing stimuli were equivalent across the schedules. Nevertheless, the stimulus-reinforcer interaction was maintained, indicating that the interaction was an associative effect.     

Classical heart-rate conditioning in the rat: The influence of curare and various setting operations

Conflicting reports concerning the success of classical conditioning of heart rate (HR) in curarized animals led to an experiment using a transfer design in which rats were classically conditioned either under curarized or restrained conditions. These conditions were then reversed for each group, giving a Curarized-Restrained (C-R) and a Restrained-Curarized (R-C) group. Results indicated that curare inhibits the conditional and unconditional HR response both during initial acquisition and after asymptotic levels of a HR CR have been attained. In addition, the C-R group did not acquire a CR during their later non-curarized restrained conditioning sessions, implying an inhibitory transfer from their earlier conditioning under curare. The UCRs during this period were normal for restrained rats. Analogous experimental“setting operations” prior to restrained conditioning were then explored by a second experiment which attempted to replicate the inhibitory transfer effect. The experimental operations investigated included: (1) restrained conditioning to investigate conditioned inhibition possibilities; (2) pseudo-conditioning types of randomized CS and UCS non-paired presentations to investigate associative vs. non-associative explanations; (3) CS-Only presentations to investigate possibilities that curare creates a blockage to the UCS, thus creating an inhibiting habituation to the CS; and (4) a group receiving curarization sessions, but no conditioning, to test the drug-only transfer effects. Results from the first and second experiments together indicated no inhibitory transfer effects under any prior experimental conditions except for those animals receiving classical conditioning while curarized. Only the CS-O group demonstrated any other kind of significant transfer effect; in this case a positive transfer accelerating later conditioning due to prior CS habituation sessions. These findings were discussed within the context of existing reports of curarized conditioning and setting operation effects on later conditioning, and various potential explanations and interpretations were explored.     

Hippocampal and cerebellar single-unit activity during delay and trace eyeblink conditioning in the rat

In delay eyeblink conditioning, the CS overlaps with the US and only a brainstem-cerebellar circuit is necessary for learning. In trace eyeblink conditioning, the CS ends before the US is delivered and several forebrain structures, including the hippocampus, are required for learning, in addition to a brainstem-cerebellar circuit. The interstimulus interval (ISI) between CS onset and US onset is perhaps the most important factor in classical conditioning, but studies comparing delay and trace conditioning have typically not matched these procedures in this crucial factor, so it is often difficult to determine whether results are due to differences between delay and trace or to differences in ISI. In the current study, we employed a 580-ms CS-US interval for both delay and trace conditioning and compared hippocampal CA1 activity and cerebellar interpositus nucleus activity in order to determine whether a unique signature of trace conditioning exists in patterns of single-unit activity in either structure. Long-Evans rats were chronically implanted in either CA1 or interpositus with microwire electrodes and underwent either delay eyeblink conditioning, or trace eyeblink conditioning with a 300-ms trace period between CS offset and US onset. On trials with a CR in delay conditioning, CA1 pyramidal cells showed increases in activation (relative to a pre-CS baseline) during the CS-US period in sessions 1-4 that was attenuated by sessions 5-6. In contrast, on trials with a CR in trace conditioning, CA1 pyramidal cells did not show increases in activation during the CS-US period until sessions 5-6. In sessions 5-6, increases in activation were present only to the CS and not during the trace period. For rats with interpositus electrodes, activation of interpositus neurons on CR trials was present in all sessions in both delay and trace conditioning. However, activation was greater in trace compared to delay conditioning in the first half of the CS-US interval (during the trace CS) during early sessions of conditioning and, in later sessions of conditioning, activation was greater in the second half of the CS-US interval (during the trace interval). These results suggest that the pattern of hippocampal activation that differentiates trace from delay eyeblink conditioning is a slow buildup of activation to the CS, possibly representing encoding of CS duration or discrimination of the CS from the background context. Interpositus nucleus neurons show strong modeling of the eyeblink CR regardless of paradigm but show a changing pattern across conditioning that may be due to the necessary contributions of forebrain processing to trace conditioning.     

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.     

Amphetamine, conditioned stimulus, and nondebilitating preshock effects on activity and avoidance: further evidence for interactions between associative and nonassociative changes

A literature survey and preliminary experiments with rats on the consequences of shock preexposure on subsequent activity and escape or avoidance showed the need for further work on the interactions between nondebilitating preshock and various test and treatment factors. The two main experiments used 16 preexposure conditions, namely, presence or absence of unavoidable punishment (36 shocks of 2.5 mA and 5 sec subdivided in three daily sessions), a light CS, a central partition in the shuttle-box, and dl-amphetamine sulfate (1 mg/kg ip 15 min before each session). In both experiments the four factors studied exerted more than additive effects on activity in preexposure sessions, leading to a very high frequency of crossing in the CS-shock-no-partition-drug condition. Upon retesting for activity (Experiment 1) suppression of locomotion by prior shock was less marked in animals preexposed to CS-US pairings in the absence of partition, while proactive amphetamine effects consisted mainly of a progressive increase of activity over successive retest sessions in the groups not preshocked. Upon retesting for light-cued, two-way avoidance acquisition (Experiment 2) the groups preexposed to US only were mostly retarded, while those preexposed to paired CS and US were mostly facilitated. Other changes, including drug pretreatment consequences, were negligible or unsystematic, but in general the data showed that the effects of various preexposure conditions on activity could not account for those on avoidance. Overall, it appears that the interactions between nondebilitating preshock and other test and treatment factors can be further exploited to clarify the respective roles of various associative and nonassociative mechanisms in modulation of activity and adaptive responding in aversive situations.     

Long-term consolidation and retention of learning-induced tuning plasticity in the auditory cortex of the guinea pig.

The major goal of this study was to determine whether classical conditioning produces long-term neural consolidation of frequency tuning plasticity in the auditory cortex. Local field potentials (LFPs) were obtained from chronically implanted adult male Hartley guinea pigs that were divided into conditioning (n = 4) and sensitization control (n = 3) groups. Tuning functions were determined in awake subjects for average LFPs (approximately 0.4 to 36.0 kHz, -20 to 80 dB) immediately before training as well as 1 h and 1, 3, 7, and 10 days after training; sensitization subjects did not have a 10-day retention test. Conditioning consisted of a single session of 30 to 45 trials of a 6-s tone (CS, 70 dB) that was not the best frequency (BF, peak of a tuning curve), followed by a brief leg shock (US) at CS offset. Sensitization control animals received the same density of CS and US presentations unpaired. Heart rate recordings showed that the conditioning group developed conditioned bradycardia, whereas the sensitization control group did not. Local field potentials in the conditioning group, but not in the sensitization group, developed tuning plasticity. The ratio of responses to the CS frequency versus the BF were increased 1 h after training, and this increase was retained for the 10-day period of the study. Both tuning plasticity and retention were observed across stimulus levels (10-80 dB). Most noteworthy, tuning plasticity exhibited consolidation (i.e., developed greater CS-specific effects across retention periods), attaining asymptote at 3 days. The findings indicate that LFPs in the auditory cortex have three cardinal features of behavioral memory: associative tuning plasticity, long-term retention, and long-term consolidation. Potential cellular and subcellular mechanisms of LFP tuning plasticity and long-term consolidation are discussed.     

Learning strategies during fear conditioning

This paper describes a model of fear learning, in which subjects have an option of behavioral responses to impending social defeat. The model generates two types of learning: social avoidance and classical conditioning, dependent upon (1) escape from or (2) social subordination to an aggressor. We hypothesized that social stress provides the impetus as well as the necessary information to stimulate dichotomous goal-oriented learning. Specialized tanks were constructed to subject rainbow trout to a conditioning paradigm where the conditioned stimulus (CS) is cessation of tank water flow (water off) and the unconditioned stimulus (US) is social aggression from a larger conspecific. Following seven daily CS/US pairings, approximately half of the test fish learned to consistently escape the aggression to a neutral chamber through a small escape hole available only during the interaction. The learning curve for escaping fish was dramatic, with an 1100% improvement in escape time over 7 days. Fish that did not escape exhibited a 400% increase in plasma cortisol and altered brain monoamine response to presentation of the CS alone. Elevated plasma cortisol levels represent classical fear conditioning in non-escaping fish, while a lack of fear conditioning and a decreased latency to escape over the training period in escapers indicates learned escape.     

Acquisition and discrimination of appetitively and aversively conditioned heart rate responses in rhesus monkeys

This report describes the acquisition of a conditional heart rate response to both classical aversive and appetitive conditioning in eight rhesus monkeys(Macaca mulatta). The behavioral paradigm consisted of two discrete one minute tones followed by the corresponding unconditional stimuli,i.e., electric shock or Purina monkey chow. A third tone followed by neither food nor shock served as a discriminative stimulus (DS). The conditional heart rate responses developed in two phases. The first phase was characterized by similar responses to both conditional stimuli and the DS. Control heart rate rose during this period. During the second phase, control heart rate decreased (five animals), the DS response disappeared, and different conditional heart rate patterns to food and shock emerged. The existence of distinct conditional response patterns indicates probable differences in the autonomic nervous regulation of the heart during aversive and appetitive conditioning.     

Associative representational plasticity in the auditory cortex: a synthesis of two disciplines

Historically, sensory systems have been largely ignored as potential loci of information storage in the neurobiology of learning and memory. They continued to be relegated to the role of "sensory analyzers" despite consistent findings of associatively induced enhancement of responses in primary sensory cortices to behaviorally important signal stimuli, such as conditioned stimuli (CS), during classical conditioning. This disregard may have been promoted by the fact that the brain was interrogated using only one or two stimuli, e.g., a CS(+) sometimes with a CS(-), providing little insight into the specificity of neural plasticity. This review describes a novel approach that synthesizes the basic experimental designs of the experimental psychology of learning with that of sensory neurophysiology. By probing the brain with a large stimulus set before and after learning, this unified method has revealed that associative processes produce highly specific changes in the receptive fields of cells in the primary auditory cortex (A1). This associative representational plasticity (ARP) selectively facilitates responses to tonal CSs at the expense of other frequencies, producing tuning shifts toward and to the CS and expanded representation of CS frequencies in the tonotopic map of A1. ARPs have the major characteristics of associative memory: They are highly specific, discriminative, rapidly acquired, exhibit consolidation over hours and days, and can be retained indefinitely. Evidence to date suggests that ARPs encode the level of acquired behavioral importance of stimuli. The nucleus basalis cholinergic system is sufficient both for the induction of ARPs and the induction of specific auditory memory. Investigation of ARPs has attracted workers with diverse backgrounds, often resulting in behavioral approaches that yield data that are difficult to interpret. The advantages of studying associative representational plasticity are emphasized, as is the need for greater behavioral sophistication.     

Signaled avoidance in the eye withdrawal reflex of the green crab

Learning in a signaled avoidance procedure was studied in the eye withdrawal reflex of the green crab, Carcinus maenas. A puff of air to the eye, which causes eye retraction, was used as the unconditioned stimulus (US). A mild vibration on the carapace, which has no effect on untrained animals, was used as a warning (conditioned) stimulus (CS). Eye withdrawal during the CS led to the omission of the otherwise scheduled US. Acquisition was rapid, reaching about 75% avoidance after 30 trials. Extinction occurred slowly over the course of 40 CS-only trials. Yoked controls did not perform as well. The behavior of experimental animals in the avoidance procedure was found to be essentially identical to the performance of animals subjected to a classical conditioning paradigm in which CS responses had no effect on US presentation. Additional groups of animals were subjected to experiments in which (a) avoidance conditioning (60 trials) was followed by classical conditioning (40 trials) or (b) classical conditioning was followed by avoidance. The behavior of these groups was, again, essentially identical. The results suggest that there may be an underlying Pavlovian mechanism for the learned response, although the contribution of an operant process is not excluded. The results expand the range of invertebrate animals in which fundamental conditioning phenomena can be demonstrated, and may provide a neuronal model for learning in a signaled avoidance procedure.     

Pavlovian conditioning of shock-elicited aggression: a discrimination procedure

Two auditory stimuli, separated by a fixed intertrial interval, were alternately presented to two rats in a closed environment. The positive conditioned stimulus (CS+) terminated with the offset of a 2-mA, 0.75-sec shock. The negative conditioned stimulus (CS-) terminated without shock. The incidence of the "stereotyped fighting posture" was recorded during the CS+, the CS-, the intertrial interval, and shock. The results showed an increase in the percentage of conditioned responses during the CS+, and a decrease during both the CS- and the intertrial interval, when the duration of the conditioned stimuli and the intertrial interval was 16 sec. Appropriate changes in the incidence of aggression during the two stimuli were obtained following the reversal of the stimulus functions. During the acquisition and reversal phases there was a between-session decrement and a within-session improvement in the incidence of aggression during the CS+, defined as warm-up. The presentation of free shocks before the conditioning sessions was effective in reducing the warm-up only when the interval between shocks was 64 sec. These data were interpreted as demonstrating classical conditioning of shock-elicited aggression, with little chance of non-associative factors contributing to the measurement of the conditioned response.