Conditioned Stimulus-unconditioned Stimulus Research Articles

Pavlovian autoshaping procedures increase plasma corticosterone levels in rats

Pavlovian autoshaping conditioned responses (CRs) are complex sequences of conditioned stimulus (CS)-directed skeletal-motor responses that are elicited by CS objects predictive of food unconditioned stimulus (US). Autoshaping CRs are observed under conditions known to be conducive to elevations in plasma corticosterone levels, as, for example, in response to the eating of food as well as in response to signals predictive of food. Two experiments investigated the relationships between Pavlovian autoshaping procedures, the performance of Pavlovian autoshaping CRs, and plasma corticosterone levels in male Long–Evans rats. In Experiment 1, rats in the CS–US paired group ( n=30) were given 20 daily sessions of Pavlovian autoshaping training wherein the insertion of a retractable lever CS was followed by the response-independent presentation of the food US. Tail blood samples obtained after the 20th autoshaping session revealed higher plasma corticosterone levels in the CS–US paired group than in the CS–US random control group ( n=10). In Experiment 2, rats ( n=35) were assessed for basal plasma corticosterone levels 2 weeks prior to autoshaping training. Plasma samples obtained immediately following the first autoshaping session, and prior to the acquisition of lever-press autoshaping CR performance, revealed higher plasma corticosterone levels in the CS–US paired group ( n=24) relative to basal levels. This effect was not observed in the CS–US random control group ( n=11). Data suggest that corticosterone release is a physiological endocrine Pavlovian CR induced by lever CS–food US pairings during Pavlovian autoshaping procedures, rather than a by-product of autoshaping CR performance. Implications of the link between autoshaping procedures and corticosterone release are discussed.

Impairment of eyeblink conditioning in GluRdelta2-mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli.

Mice lacking the glutamate receptor subunit delta2 (GluRdelta2) are deficient in cerebellar long-term depression (LTD) at the parallel fibre-Purkinje cell synapses. We conducted delay and trace eyeblink conditioning with these mice, using various temporal intervals between the conditioned stimulus (CS) and unconditioned stimulus (US). During trace conditioning in which a stimulus-free trace interval (TI) of 250, 100 or 50 ms intervened between the 352-ms tone CS and 100-ms US, GluRdelta2-mutant mice learned as successfully as wild-type mice. Even in the paradigm with TI = 0 ms, in which the end of CS and onset of US are simultaneous, there was no difference between the GluRdelta2-mutant and wild-type mice in their acquisition of a conditioned response. However, in the delay paradigm in which the 452-ms CS overlapped temporally with the coterminating 100-ms US, GluRdelta2-mutant mice exhibited severe learning impairment. The present study together with our previous work [Kishimoto, Y., Kawahara, S., Suzuki, M., Mori, H., Mishina, M. & Kirino, Y. (2001) Eur. J. Neurosci., 13, 1249-1254], indicates that cerebellar LTD-independent learning is possible in paradigms without temporal overlap between the CS and US. On the other hand, GluRdelta2 and cerebellar LTD are essential for learning when there is CS-US temporal overlap, suggesting that the cerebellar neural substrates underlying eyeblink conditioning may change, depending on the temporal overlap of the CS and US.

Patterns of Fos expression in the amygdala and ventral perirhinal cortex induced by training in an olfactory fear conditioning paradigm.

The activation of amygdaloid nuclei, the ventral perirhinal cortex (vPRh), and several other brain areas in the rat during the acquisition and expression of olfactory fear conditioning was assessed through Fos immunocytochemistry in 3 separate experiments. The results of Experiment 1 suggest that olfactory and somatosensory inputs may functionally converge in the anterior region ot the medial nucleus (aMe). The results of Experiment 2 indicate that the aMe exhibited significantly greater Fos-like immunoreactivity (FLI) in subjects acquiring conditioned stimulus-unconditioned stimulus associations than in those presented with the same olfactory and somatosensory stimuli in a manner that precluded acquisition. The results of Experiment 3 indicate that the vPRh appeared to exhibit learning-related increases in FLI during the expression of previously acquired associations. Collectively, these data suggest that the aMe and vPRh may be critically involved in different aspects of olfactory fear conditioning.

Associative learning and latent inhibition in a conditioned suppression paradigm in humans

A paradigm based on conditioned suppression of ongoing motor activity, sensitive to latent inhibition (LI), was developed and tested in healthy volunteers. Subjects were trained to move disks from one peg to another with a high degree of regularity in the Tower of Toronto puzzle, a well-known cognitive skill learning task. Once this was achieved, they were submitted to a Pavlovian conditioning procedure. The conditioned stimulus (CS) was a pure tone and the unconditioned stimulus (US) a loud white noise. The resulting response suppression was assessed by a transient increase in latency of the hand movements. In control subjects, there was non-contingent CS and US presentation. The results evidenced conditioning after a single CS–US pairing. Following five preexposures to the to-be-conditioned CS, however, conditioning was abolished, seemingly expressing LI. Because a weak unconditioned response to the tone was observed after its first two presentations, an additional experiment was performed with two preexposures to the to-be-conditioned CS. With such procedure, conditioning was obtained, supporting the existence of LI in the preceding experiment. These results indicate that the present paradigm may be useful for the study of LI in human subjects, having the advantage of being similar to the experimental conditions used in the majority of LI studies in experimental animals.

The basolateral amygdala complex is involved with, but is not necessary for, rapid acquisition of Pavlovian 'fear conditioning'.

A major hypothesis about lateral/basolateral amygdala complex (BLC) function in memory proposes that the BLC is the site where conditioned stimulus-unconditioned stimulus (CS-US) associations are formed and permanently stored during Pavlovian 'fear conditioning.' Thus, according to this hypothesis, the BLC is necessary for the acquisition and expression of both discrete-cue and contextual Pavlovian fear conditioning. This hypothesis clearly requires that animals with complete lesions of the BLC be completely unable to acquire Pavlovian fear conditioning. In this experiment, distribution of training and testing trials over three sessions revealed that rats with complete BLC lesions rapidly acquired a contextual CS-US association (as assessed with freezing behaviour), although their performance, as expected, did not equal that of sham operated controls. Irrespective of the nature of the freezing deficit relative to controls, the learning in the BLC-lesioned rats strongly indicates that Pavlovian fear conditioning CS-US associations can be rapidly acquired in the absence of the BLC, and that the BLC cannot therefore be necessary for their acquisition.

Classically conditioned responses following repeated insulin and glucose administration in humans

This paper describes the neural basis and the role of Pavlovian conditioning in the modification of blood glucose and related endocrine parameters after repeated insulin and glucose administration. Pavlovian conditioning requires that conditioned stimulus (CS) and unconditioned stimulus (US) are both detected in the central nervous system (CNS), where the CS–US association takes place. We will therefore elucidate the detectability of insulin and glucose in the CNS. Since current data focus almost exclusively on animals, we conducted a placebo-controlled insulin conditioning experiment in humans (Experiment 1). Compared with the control group with CS-placebo pairings throughout, the experimental group with previous CS-insulin pairings in the acquisition phase showed a conditioned decrease in blood glucose and a trend for a conditioned baseline insulin increase, and an increase in cortisol levels relative to baseline and cumulative number of neuroglycopenic symptoms in the CS-placebo test session. The conditionability of glucose administration also had to be examined; experiments using an arbitrary CS and glucose are extremely rare, even in animals. Glucose is the natural stimulus for endogenous insulin secretion, so studies on cephalic-phase insulin release (CPIR) will be reviewed in this paper. We implemented a placebo-controlled three-group design (Experiment 2): Subjects received either CS-insulin, CS-glucose or CS-placebo pairings during the acquisition. Together, our results demonstrate the conditionability mainly of insulin, but also of glucose effects in healthy humans. The clinical relevance and future research perspectives are outlined with an emphasis on insulin in the brain and its role in learning and memory.

Short-lasting conditioned stimulus applied to the middle cerebellar peduncle elicits delayed conditioned eye blink responses in the decerebrate ferret.

In delay eye blink conditioning, the conditioned stimulus (CS) ends at the time of the unconditioned stimulus (US). If the CS duration is decreased, there will be a 'trace' period with no ongoing CS before the onset of the US. During this period some neural activity has to continue after the CS offset to: (i) permit association between the CS and the US; and (ii) elicit a conditioned response appearing after the CS offset. In this study we test the role of the cerebellum in maintaining CS activity required for eliciting a conditioned response after the CS offset. Decerebrate ferrets were trained in a delay conditioning paradigm with an electrical stimulation of the forelimb as CS and of the periorbital area as US. The conditioned responses in the upper eyelid were monitored with electromyographical techniques. In well-trained animals, test CSs of short duration down to 0.2 ms were applied to the forelimb or the middle cerebellar peduncle, while the interstimulus interval between CS onset and US onset was kept constant at 300 ms. Test CSs of short duration applied to the forelimb elicited conditioned responses. More importantly, also a short-lasting CS to the middle cerebellar peduncle could elicit conditioned responses. The results indicate that precerebellar CS pathways are not required for maintaining the neural activity that elicits conditioned responses after the CS offset. It is suggested that neurons maintaining such activity are located in the cerebellum, either the cortex alone or the cortex and the deep nuclei.

Lesions of the Intermediate Medial Hyperstriatum Ventrale Impair Sickness-Conditioned Learning in Day-Old Chicks

Lesion studies show that the intermediate medial hyperstriatum ventrale (IMHV), a forebrain visual association area in chicks, is involved in learning and memory for one-trial passive avoidance and imprinting. We examined the effects of IMHV lesions in a one-trial, nongustatory, sickness-conditioned learning task. This task is similar to passive avoidance and imprinting because all three tasks require the chick to remember visual cues in order to respond correctly. However, sickness-conditioned learning differs from imprinting and passive avoidance because it uses sickness as the aversive stimulus and there is a longer conditioned stimulus-unconditioned stimulus interval (30-min delay compared to seconds). Bilateral IMHV lesions given 24 h before training impaired the ability of the chicks to avoid a bead associated with sickness produced by lithium chloride injection, as did pretraining unilateral left or right IMHV lesions. Post-training IMHV lesions given 1 h after training did not impair avoidance of the test bead in the sickness-conditioned learning task. However, lesioned chicks showed generalized avoidance of all test beads. The pretraining lesion results are similar to those found in imprinting and passive avoidance learning; however, the effects of unilateral IMHV lesions differed. Post-training lesion effects are similar to those found in passive avoidance learning. We propose that both left and right IMHV are necessary for sickness-conditioned learning and that post-training IMHV lesions impair the ability of the chick to learn or remember the association between the color of the bead and the aversive consequences of LiCl injection.

Ketaset–Rompun Extends the Temporal Gradient for Hypothermia-Induced Retrograde Amnesia

In studies of experimentally induced retrograde amnesia (RA), as the interval between training and the amnestic treatment is lengthened, amnesia decreases (4). This temporal gradient for RA has been reported with a wide variety of amnestic agents, including RA produced by thermoregulatory disturbances (8). This temporal gradient for RA is not unlike certain characteristics of classical conditioning, where weaker conditioned responding occurs when the interval between the conditioned stimulus (CS) and unconditioned stimulus (US) is lengthened. Furthermore, there is evidence that administration of anesthetics can lengthen the “effective conditioning” interval between the CS and US, as demonstrated in a conditioned taste-aversion (CTA) procedure (10). In that study, little conditioning was observed when a 3-h delay (or more) was incorporated between presentations of the CS (flavor) and the US (toxin). However, if subjects were anesthetized immediately after the CS was delivered and remained anesthetized during the CS–US interval, strong conditioning was observed with CS–US intervals of up to 9 h. The aim of the present experiment was to determine if the temporal gradient for hypothermia-induced RA could also be lengthened. That is, we tested whether the interval between training and hypothermia treatment could be lengthened by anesthetizing subjects with Ketaset–Rompun. The results indicate that the training-to-amnestic agent interval could be lengthened within moderate limits. The implications for hypothermia-induced RA is further discussed.

Nicotine enhancement of contextual fear conditioning

Nicotine has been suggested to have cognitive enhancing effects. The present study examined the effects of nicotine and the nicotinic antagonist mecamylamine on contextual fear conditioning in C57BL/6 mice. The fear conditioning task was chosen because the task examines two types of learning: contextual learning, and conditioned stimulus (CS)–unconditioned stimulus (US) learning. Multiple doses of nicotine were tested and 0.5 mg/kg nicotine, given on both training and testing days, improved contextual learning but had no effect on formation of an auditory CS–US association. No effect was found at lower doses or when nicotine was given on training day only, or testing day only. The nicotinic receptor antagonist mecamylamine (1 and 2 mg/kg) did not alter contextual fear conditioning but mecamylamine did prevent the nicotine-associated increase in contextual learning. A higher dose of nicotine (1 mg/kg, training day only) interfered with contextual conditioning when the context was paired with both the CS and US, but had no effect on the auditory CS–US association. This effect of 1 mg/kg nicotine on contextual learning disappeared when mice were trained without the CS. The present results indicate that nicotine enhancement of contextual fear conditioning is dose-dependent, but the presence of nicotine is required both during training and testing.

Savings after extinction in conditioning of the rabbit’s nictitating membrane response

The present experiments examined two forms of savings after the extinction of a conditioned response (CR) in the rabbit nictitating membrane preparation. Each experiment entailed three groups. First, a reacquisition (RAQ) group received initial conditioned stimulus-unconditioned stimulus (CS—US) acquisition, followed by CS-alone extinction, and then CS—US reacquisition with the same stimulus. Second, a cross-modal acquisition (CMA) group received initial acquisition and extinction with one stimulus but received its final stage of training with a stimulus from another modality. Third, a naive acquisition group (designated as REST) received only the final stage of CS—US training. In all three groups, tone and light were used as CSs in a counterbalanced fashion. In addition, the experiments tested whether the manipulation of contextual factors—namely, trial spacing and the number of trials per session—could influence the rates of acquisition in Groups RAQ and CMA after extinction. The experiments demonstrated that (1) reacquisition of the CR to the original CS was very rapid relative to Group REST, (2) cross-modal acquisition was also rapid relative to Group REST, and (3) in Group CMA, tests of the original CS showed concurrent recovery, which is a new phenomenon and is distinct from spontaneous recovery. However, the relative rates of acquisition after extinction appeared constant across the contextual manipulations. The results are discussed with respect to the relative stability of excitatory and inhibitory learning, as well as two alternative theories of savings after extinction.

Drosophila Conditioned Courtship: Two Ways of Testing Memory

In Drosophila, courtship reduction in male flies that have previous experience of courting a mated female is a result of the counterconditioning of an attractive unconditioned stimulus (US)--the aphrodisiac--which becomes an aversive conditioned stimulus (CS) after being paired with an aversive US--the antiaphrodisiac. In a retention test with a virgin female lacking the antiaphrodisiac, males retain a lower level of courtship for 3 hr after training. However, a measure of courtship suppression, the learning index (LI), decreases significantly after only 1 hr. In contrast, in the retraining test with a mated female, the LI shows no decrease for 8 hr but falls below significance 16 hr after training. These results are discussed in terms of the transfer of training. Nonspecific transfer and nonassociative behavioral modifications play little, if any, role in the transfer of training. The retraining test is recommended as a new protocol for studying conditioned courtship. According to the model proposed here, in tests with a virgin female, the duration of memory retention is limited by the retention of the direct association between the CS and the aversive motivational system or by the retention of an internal representation of the US. In retraining tests, the CS-US association seems to be the only factor involved in transfer 3 or more hours after training.

Relationships among age, conditioned stimulus-unconditioned stimulus interval, and neuropsychological test performance.

To evaluate the effect of age at various conditioned stimulus (CS)-unconditioned stimulus (US) intervals, 144 young, middle-aged, and older adults were tested on eyeblink classical conditioning at CS-US intervals of 500, 1,000, or 1,500 ms. Reaction time, response timing, motor learning, declarative memory, and attention were assessed to identify correlates of conditioning at various CS-US intervals. Previously reported middle-aged and older adults were impaired at a 400-ms CS-US interval, but the addition of 100 ms to the CS-US interval in this study enabled equal conditioning in middle-aged and young adults. At a 1,000-ms CS-US interval, older adults remained significantly impaired. It was only at the 1,500-ms CS-US interval that conditioning was equal for the 3 age groups. Measures of reaction time, timing, and motor learning were not correlated systematically with conditioning. Whereas the results of age differences at various CS-US intervals were clear and striking, patterns of relationships among neuropsychological and conditioning variables were not consistent in indicating sources of age differences.

Inhibition of the mesoamygdala dopaminergic pathway impairs the retrieval of conditioned fear associations.

Previous findings have demonstrated that systemic dopaminergic manipulations impair the retrieval of Pavlovian conditioned fear. A second-order fear-conditioning paradigm was used to test whether the dopaminergic projection from the ventral tegmental area (VTA) to the lateral and basal amygdala (LBA) can affect conditioned fear. Phase 1 entailed conditioned stimulus-unconditioned stimulus (CS1-US) pairings. In Phase 2, drugs were infused in either the LBA or VTA prior to pairings of CS2 (a second cue) with CS1. In Phase 3, freezing behavior elicited by CS2 was tested without drugs. Infusions of the D2 agonist quinpirole into the VTA or of the D1 antagonist SCH 23390 into the LBA caused a decrease in freezing to CS2. Both manipulations decrease D1 receptor activation in the LBA. Infusions of the D1 agonist SKF 38393 into the LBA had no effect. This pattern of results is consistent with the hypothesis that the VTA-LBA dopaminergic projection modulates the retrieval of an association between a CS and footshock US.

Acquisition of fear conditioning in rats requires the synthesis of mRNA in the amygdala.

In this study, the role of mRNA synthesis in the amygdala was studied during the acquisition of conditional fear. Rats with cannulas placed in the basolateral region of the amygdala were trained with a series of noise-shock pairings in a distinctive observation chamber. One half of the rats were pretreated with the mRNA synthesis inhibitor actinomycin-D (act-D). Responding to the training context and the auditory stimulus in a novel context measured by defensive freezing was assessed. Pretreatment with act-D significantly attenuated fear responses to both stimuli. Animals receiving act-D injections exhibited normal reactions to the conditioned stimulus-unconditioned stimulus pairings in the initial training session and displayed normal learning when retrained 7 days after injections. These results indicate that the transcription of new mRNA and subsequent protein synthesis in the amygdala may be essential for neural plasticity during this form of associative learning.

Sequence-dependent interactions between transient calcium and transmitter stimuli in activation of mammalian brain adenylyl cyclase

Recent evidence implicates Ca 2+/CaM-sensitive adenylyl cyclase (AC) as a molecular coincidence detector for temporally paired stimuli during associative learning. During conditioning in Aplysia, AC is optimally activated when Ca 2+ influx, the cellular signal for the conditioned stimulus (CS), precedes binding of modulatory transmitter, the cellular signal for the unconditioned stimulus (US). This sequence preference of the AC for Ca 2+-before-transmitter, parallels the CS-preceding-US pairing requirement of classical conditioning. In this study, we have examined the response of AC from rat cerebellum to brief exposures to Ca 2+ and to transmitter in a perfused membrane assay. We observed modest synergism between Ca 2+ and transmitter in activating AC. Activation was more effective when a Ca 2+ stimulus immediately preceded a transmitter stimulus than when the two stimuli were delivered in the reverse order. Thus, rat cerebellar AC displayed a sequence preference for optimal activation by paired stimuli similar to that observed in Aplysia; this sequence dependence could contribute to the CS–US sequence requirement observed in most mammalian classical conditioning paradigms.

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.

Amount of training affects associatively-activated event representation

The effects of variations in the amount of training on behavioral plasticity were examined in three experiments that used appetitive Pavlovian conditioning procedures with rats. In experiments 1 and 2, an auditory conditioned stimulus (CS) substituted for a food unconditioned stimulus (US) in the acquisition of new learning about the food US after small numbers of CS–US pairings, but not after larger numbers of pairings. After limited exposure to the relation between the auditory CS and food, pairings of the CS with the toxin LiCl, in the absence of food, were sufficient to establish an aversion to the food US that was previously paired with that CS. This CS-mediated learning did not occur after more extensive exposure to the CS–food relation. In contrast, in experiment 3, mediated performance of previously-established conditioned responding was unaffected by the number of CS–US pairings used to establish that performance. Conditioned responding to the auditory CS remained sensitive to post-training devaluation of the food US regardless of the amount of initial CS–US training. Implications of these results for the investigations of cortical and other brain mechanisms of behavioral plasticity are discussed.

Conducting exposure treatment in multiple contexts can prevent relapse

The acquisition of anxiety disorders (e.g., phobias) is often thought to be mediated by classical conditioning processes (e.g., Wolpe, 1958, Psychotherapy by reciprocal inhibition Wolpe and Rowan, 1989, Behaviour Research and Therapy, 27, 583–585). Thus, the success of exposure therapy is possibly a consequence of extinction, and factors affecting extinction in Pavlovian conditioning are potentially relevant to clinicians who administer exposure therapy. The present experiments investigated the effects of conducting extinction in multiple contexts using rats as subjects in a conditioned suppression paradigm. In Experiment 1, subjects received conditioned stimulus (CS) and unconditioned stimulus (US) pairings in one context followed by extinction of that CS in one or three other contexts. When tested in an associatively neutral context (i.e., different from those of conditioning or extinction), rats that had received extinction in three contexts exhibited less responding to the CS than rats that had received extinction in one context. In Experiment 2, CS–US training occurred in either one or three contexts, followed by extinction of that CS in three other contexts. Testing in a neutral context revealed that rats conditioned in multiple contexts showed greater responding to the CS than rats trained in a single context. The results are discussed in the framework of memory retrieval, and the clinical implications are explored.

Classical eyeblink conditioning in two strains of mice: conditioned responses, sensitization, and spontaneous eyeblinks.

Conditioned eyeblink responses (CRs), sensitization, and spontaneous eyeblinks were studied in C57BL/6J and BALB/c mice. Both strains of mice acquired CRs during 10 days of classical delay eyeblink conditioning. The BALB/c mice reached a higher asymptotic CR level than the C57BL/6J mice. The CRs were extinguished and recovered in both strains following conditioned stimulus-alone and paired conditioned stimulus-unconditioned stimulus training. During 10 days of explicitly unpaired training, the control groups showed no signs of sensitization and low incidence of spontaneous eyeblinks. When switched to paired training, the unpaired groups exhibited significant conditioned inhibition. These results suggest that strain differences must be considered in experimental design and data interpretation for these basic aspects of associative learning and memory.