Hippocampal theta (3-8Hz) activity during classical eyeblink conditioning in rabbits

In 1978, Berry and Thompson showed that the amount of theta (3–8 Hz) activity in the spontaneous hippocampal EEG predicted learning rate in subsequent eyeblink conditioning in rabbits. More recently, the absence of theta activity during the training trial has been shown to have a detrimental effect on learning rate. Here, we aimed to further explore the relationship between theta activity and classical eyeblink conditioning by determining how the relative power of hippocampal theta activity [theta/(theta + delta) ratio] changes during both unpaired control and paired training phases. We found that animals with a higher hippocampal theta ratio immediately before conditioning learned faster and also that in these animals the theta ratio was higher throughout both experimental phases. In fact, while the hippocampal theta ratio remained stable in the fast learners as a function of training, it decreased in the slow learners already during unpaired training. In addition, the presence of hippocampal theta activity enhanced the hippocampal model of the conditioned response (CR) and seemed to be beneficial for CR performance in terms of peak latency during conditioning, but did not have any effect when the animals showed asymptotic learning. Together with earlier findings, these results imply that the behavioral state in which hippocampal theta activity is absent is detrimental for learning, and that the behavioral state in which hippocampal theta activity dominates is beneficial for learning, at least before a well-learned state is achieved.     

Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: a dose-response study in the rat

The behavioral analysis of laboratory rats is usually confined to the level of overt behavior, like locomotion, behavioral inhibition, instrumental responses, and others. Apart from such visible outcome, however, behaviorally relevant information can also be obtained when analyzing the animals' ultrasonic vocalization, which is typically emitted in highly motivational situations, like 22-kHz calls in response to acute or conditioned threat. To further investigate such vocalizations and their relationship with overt behavior, we tested male Wistar rats in a paradigm of Pavlovian fear conditioning, where a tone stimulus (CS) was preceding an aversive foot-shock (US) in a distinct environment. Importantly, the shock dose was varied between groups (0-1.1 mA), and its acute and conditioned outcome were determined. The analysis of visible behavior confirms the usefulness of immobility as a measure of fear conditioning, especially when higher shock doses were used. Rearing and grooming, on the other hand, were more useful to detect conditioned effects with lower shock levels. Ultrasonic vocalization occurred less consistently than changes in overt behavior; however, dose-response relationships were also observed during the phase of conditioning, for example, in latency, call rate and lengths, intervals between calls, and sound amplitude. Furthermore, total calling time (and rate) were highly correlated with overt behavior, namely behavioral inhibition as measured through immobility. These correlations were observed during the phase of fear conditioning, and the subsequent tests. Importantly, conditioned effects in overt behavior were observed, both, to the context and to the CS presented in this context, whereas conditioned vocalization to the context was not observed (except for one rat). In support and extent of previous results, the present data show that a detailed analysis of ultrasonic vocalization can substantially broaden and refine the spectrum of analysis in behavioral work with rats, since it can provide information about situational-, state-, and subject-dependent factors which are partly distinct from what is visible to the experimenter.     

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 conditioned inhibition of fear during shuttlebox avoidance behavior

Three experiments explored the development and function of conditioned inhibition of fear during the acquisition and maintenance of shuttlebox avoidance behavior. The development of inhibition to an exteroceptive feedback stimulus was found to be a function of the number of successive avoidance responses to which the animal had been trained and of the duration of the intertrial interval, a parameter shown also to affect the rate of acquisition of avoidance learning. Master animals who learned the instrumental avoidance response, and yoked animals who did not, showed equivalent inhibitory fear conditioning in each experiment. The results of one experiment suggest that conditioned inhibition plays no important role in “protecting” fear conditioned to the discrete warning signal during avoidance maintenance. These data indicate that feedback stimuli develop their inhibitory properties by a Pavlovian process and that certain aspects of their function may, therefore, be readily understood within the framework of mediational two-process learning theory.     

Goal-tracking behavior in the pond snail, Lymnaea stagnalis

The occurrence of goal-tracking, an unconditioned stimulus (US)-directed autoshaping behavior, was studied in open-field tests with control and classically conditioned pond snails, Lymnaea stagnalis. In an appetitive classical conditioning paradigm with a tactile stimulus as conditioned stimulus (CS) and a localized food stimulus as US a conditioned feeding response built up in the experimental but not in the control animals. In the post-training open-field tests the experimental group alone showed an enhanced attraction toward the source of water current in the environment which previously signalled the arrival of the US but did not act as CS in the classical conditioning procedure. We suggest that this stimulus-directed goal-tracking behavior in Lymnaea is the result of a classical-operant interaction, described so far only in vertebrate animals, and that neurophysiological analysis of this behavior is possible in this snail.     

Mediodorsal thalamic lesions impair trace eyeblink conditioning in the rabbit

Rabbits received lesions of the mediodorsal nucleus of the thalamus (MDN) or sham lesions and were subjected to classical eyeblink (EB) and heart rate (HR) conditioning. All animals received trace conditioning, with a.5-sec tone conditioned stimulus, a .5-sec trace period, and a 50-msec periorbital shock unconditioned stimulus. Animals with MDN lesions acquired the EB conditioned response (CR) more slowly than sham-lesioned animals. However, previous studies have shown that MDN damage does not affect delay conditioning using either .5-sec or 1-sec interstimulus intervals. The lesions had no significant effect on the HR CR. These results suggest that information processed by MDN and relayed to the prefrontal cortex is required for somatomotor response selection under nonoptimal learning conditions.     

Elicited versus emitted behavior: Time to abandon the distinction

The concept of emitted behavior was formulated as a part of the original argument for the validity of a new kind of learning called operant conditioning. The rationale for operant conditioning contrasted it with Pavlovian or classical conditioning, which was (and remains) fundamentally based on responses to conditioned and unconditioned stimuli. Classical conditioned responses were said to be elicited. In contrast, operant behavior was viewed as emitted and controlled primarily by response consequences rather than antecedents. I argue that the distinction between emitted and elicited behavior is no longer warranted for three major reasons. First, the distinction was based on a view of Pavlovian conditioning that is no longer viable. Second, the distinction is incompatible with both empirical data and contemporary conceptualizations of operant behavior. Third, the only way to overcome these problems is to define emitted and elicited in terms of the type of conditioning (operant and classical) that produces these behaviors, but that approach makes the definitions circular and does not avoid implications of the terms that are misleading and counterproductive in light of contemporary research and thinking.     

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.     

A comparison of neuronal reactions during classical and instrumental conditioning under similar conditions

During the elaboration of an instrumental reflex, it is not obligatory to use a conditioned stimulus, which signals the necessity to generate an instrumental reaction in order to receive reinforcement. However, the presence of a conditioned stimulus simplifies analysis of instrumental reaction, which in this case is the response to the conditioned stimulus. On the other hand, it is necessary to distinguish between instrumental and classical conditioning, since in both cases the response to a conditioned stimulus increases. We studied neuronal analogs of classical and instrumental conditioning in the identified neurons responsible for the defensive closure of the pneumostome in the Helix mollusk under the same conditions. During classical conditioning, a mollusk received punishment after a tactile stimulus. During instrumental conditioning, a mollusk received punishment when an identified neuron did not generate an action potential in response to a tactile stimulus. The appearance of a painful stimulus did not depend on the generation or failure of a spike in the related control neuron. Another tactile stimulus, which was never paired with an unconditioned stimulus, was used as a discriminated stimulus. We also compared the behavior of such identified neurons during pseudoconditioning. The experiments were carried out in a semi-intact preparation. We examined how responses to the tactile and painful stimuli changed during different forms of training. It was shown that the dynamics of neuronal responses to a conditioned tactile stimulus were much more complex during instrumental conditioning and consisted of several phases. Throughout a learning session, neural system consecutively acquired information as to which kind of learning was presented, whether a reaction of the neural system must be generated or inhibited and which instrumental reaction is correct. We have demonstrated that response to a painful stimulus during classical conditioning decreases after short-term initial increase. However, during instrumental learning, the neurons controlling instrumental action remained highly sensitive to the unconditioned stimulus. Meanwhile, foreign neurons decreased their responses to the unconditioned stimulus. We may tentatively conclude that classical and instrumental paradigms are fundamentally different at the cellular level.     

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.     

Lesions of the entorhinal cortex impair acquisition of hippocampal-dependent trace conditioning

Rabbits with the electrolytic lesions of bilateral entorhinal cortex (EC) were trained with the hippocampal-dependent trace conditioning of the nictitating membrane response. The multiple-unit activity of the hippocampal CA1 region was recorded during conditioning. The conditioned stimulus was a tone (1 kHz, 85 dB, 200-ms duration), the unconditioned stimulus was a corneal air puff (3 psi, 150-ms duration), and the interstimulus interval was 750 ms. The EC-lesioned animals showed only 30% conditioned response (CR) by the ninth session while the sham-operated animals showed above 80% CR. The lesioned animals did not show learning-related changes in the hippocampal activity. When the training was switched to the 300-ms interstimulus interval trace conditioning, both groups learned above 80% CR. The EC-lesioned animals, however, showed less learning-related activity in the hippocampus than the sham-operated animals. These results suggest that the development of the learning-related activity in the hippocampus depends on the intact EC, and that the EC may provide a possible pathway conveying learning information from the cerebellum or cerebral cortex to the hippocampus during the trace conditioning.     

Blocking of inhibitory conditioning within a serial conditioned stimulus-conditioned inhibitor compound: Maintenance of acquired behavior without an unconditioned stimulus

Well-trained classically conditioned stimuli, presented unreinforced, were protected from extinction when they were followed by a signal of the omission of the reinforcer (conditioned inhibitor Konorskian type) in eight cats. An aversive classical conditioning paradigm with shock as the reinforcer was used. Of several behavioral (leg flexion, vocalization) and organismic arousal (heart rate, respiration rate, respiration amplitude) measures of conditioned responses, the respiration amplitude changes were found to be most informative for the continuous assessment of elicited arousal of low and medium intensity. In all subjects conditioned stimuli presented during extinction in serial compound with the conditioned inhibitor elicited larger responses than did conditioned stimuli presented alone during extinction. The mechanism of protection from extinction in a paradigm in which the elicitor of learned behavior occurs prior to the conditioned inhibitor provides the organism with the mechanism for the maintenance of learned behavior in the absence of a reinforcer.     

Formulation of a behavior system for sexual conditioning

Empirical and conceptual developments that led to the formulation of a behavior system for the sexual conditioning of male Japanese quail are described. Initial efforts concentrated on conditioning with localized conditioned stimuli and on identifying behavioral indices of conditioning. Later, learning about species-typical cues and about contextual cues was also explored, and it became evident that different types of cues control different aspects of sexual behavior. The results were used to formulate a behavior system containing both response and stimulus dimensions. In this system, contextual cues and local cues are assumed to elicit only general search behavior unconditionally. In contrast, unconditioned responses to species-typical cues of a female quail include general search, focal search, and copulatory behavior. General search, focal search, and copulatory behavior can become conditioned to local cues. Conditioning can also modify focal search behavior elicited by species-typical cues and can result in various modulatory influences between different types of stimuli. The behavior system approach provides a framework for organizing the diverse sexual conditioning effects and suggests future directions for investigation.     

Timing at the Start of Associative Learning

Some theories of associative learning imply that time plays a fundamental role in the acquisition process. Consistent with these theories, this paper presents evidence that the time from the onset of a conditioned stimulus (CS) until presentation of the unconditioned stimulus (US) is learned very rapidly at the start of training. We report two autoshaping studies and a study on aversive conditioning in goldfish in which we examine timing at the start of conditioning. We also review data from a number of other conditioning preparations, including fear-potentiated startle, appetitive conditioning in rats, and eyeblink conditioning in rabbits, that report conditioned response (CR) timing early in training. Acquisition speed and the very first expressions of conditioned responding often show sensitivity to the time of US presentation. In instances where temporal control is slowly expressed, it is likely due to performance factors, not to slow learning about time. In fact, the learning about time may be a necessary condition for associative learning.     

Inactivation of the anterior cingulate cortex impairs extinction of rabbit jaw movement conditioning and prevents extinction-related inhibition of hippocampal activity

Although past research has highlighted the involvement of limbic structures such as the anterior cingulate cortex (ACC) and hippocampus in learning, few have addressed the nature of their interaction. The current study of rabbit jaw movement conditioning used a combination of reversible lesions and electrophysiology to examine the involvement of the hippocampus and the ACC during acquisition, performance, and extinction. We found that microinfusions of procaine into the ACC did not significantly alter the rate of behavioral learning or the amplitude of hippocampal conditioned unit responses, but that they disrupted the rhythmic periodicity of conditioned jaw movements. During extinction, whereas controls showed a rapid decline in behavioral CRs and active inhibition of hippocampal unit responses, ACC lesioned rabbits showed a persistence of conditioning-related hippocampal activity and behavioral responding. The results show that the ACC can be important for adaptive suppression of conditioned behavior and suggest a crucial physiological modulation of hippocampus by ACC during extinction.     

Timing of conditioned responses utilizing electrical stimulation in the region of the interpositus nucleus as a CS

A large body of evidence indicates that the cerebellum is essential for the acquisition, retention, and expression of the standard delay conditioned eyeblink response and that the basic memory trace appears to be established in the anterior interpositus nucleus (IP). Adaptive timing of the conditioned response (CR) is a prominent feature of classical conditioning—the CR peaks at the time of onset of the unconditioned stimulus (US) over a wide range of CS-US interstimulus intervals (ISI). A key issue is whether this timing is established by the cerebellar circuitry or prior to the cerebellum. In this study timing of conditioned eyeblink responses established via electrical stimulation of the interpositus nucleus as a conditioned stimulus (CS) was analyzed prior to and following modification of the CS-US interval in well-trained rabbits. Consistent with previous results, learning under these conditions is very rapid and robust. The CR peak eyeblink latencies are initially timed to the US onset and adjust accordingly to lengthening or shortening of the CS-US interval, just as with peripheral CSs. The acquisition of conditioned eyeblink responses by direct electrical stimulation of the IP as a CS thus retains temporal flexibility following shifts in the CS-US delay, as found in standard classical eyeblink conditioning procedures.     

Timing of Conditioned Responses Utilizing Electrical Stimulation in the Region of the Interpositus Nucleus as a CS

A large body of evidence indicates that the cerebellum is essential for the acquisition, retention, and expression of the standard delay conditioned eyeblink response and that the basic memory trace appears to be established in the anterior interpositus nucleus (IP). Adaptive timing of the conditioned response (CR) is a prominent feature of classical conditioning-the CR peaks at the time of onset of the unconditioned stimulus (US) over a wide range of CS-US interstimulus intervals (ISI). A key issue is whether this timing is established by the cerebellar circuitry or prior to the cerebellum. In this study timing of conditioned eyeblink responses established via electrical stimulation of the interpositus nucleus as a conditioned stimulus (CS) was analyzed prior to and following modification of the CS-US interval in well-trained rabbits. Consistent with previous results, learning under these conditions is very rapid and robust. The CR peak eyeblink latencies are initially timed to the US onset and adjust accordingly to lengthening or shortening of the CS-US interval, just as with peripheral CSs. The acquisition of conditioned eyeblink responses by direct electrical stimulation of the IP as a CS thus retains temporal flexibility following shifts in the CS-US delay, as found in standard classical eyeblink conditioning procedures.     

Deficits in acquisition and extinction of conditioned responses in mGluR7 knockout mice

Metabotropic glutamate receptor 7 (mGluR7) is expressed in brain regions implicated in emotional learning and working memory, and previous behavioral experiments indicated contributions of mGluR7 to various complex behaviors. In the present study, we investigated the specific effects of mGluR7 deletion on a variety of conditioning paradigms that model crucial neurocognitive and psychopathological behavioral phenomena. Null-mutant mGluR7^−/− mice displayed defects during scheduled appetitive conditioning, acquisition and extinction of appetitive odor conditioning, extinction of response suppression-based conditioned emotional responding (CER), acquisition of discriminative CER, and contextual fear conditioning. mGluR7^−/− animals were slower to acquire the association between a conditioned stimulus and a positive or negative reinforcer, but eventually reached similar performance levels to their wildtype littermates. Notably, extinction learning of conditioned responses was slower in mGluR7^−/− compared to wildtype animals. The observed delays in the acquisition of complicated stimulus associations across conditioning procedures may suggest a critical role for mGluR7 in neurocognitive functions and psychopathology.     

Pavlovian conditioning in human skilled motor behavior

The role of Pavlovian contingencies in human skilled motor behavior was investigated in three experiments by means of a new conditioning preparation. In Experiment 1 the present method was shown to be appropriate for the study of associative learning. Subjects who experienced a standard delay configuration performed significantly more conditioned responses than subjects who received either backward conditioning or random pairings. Stimulus generalization was shown to be slight in two additional groups. Subsequent experiments examined conditioning with multiple conditioned stimuli (CSs). In particular, in Experiment 2 some reciprocal overshadowing was demonstrated when two conditional stimuli (tone and vibration) were compounded. Experiment 3 investigated blocking. Blocking was less than expected, however. Subjects’ perceptions of the stimuli and reaction time data suggest that a certain proportion had shifted their attention to the added element of the CS compound. Results are discussed in relation to other studies on Pavlovian learning in humans and animals, which are concerned with “stimulus selection.”     

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.