Trace and delay eyeblink conditioning: contrasting phenomena of declarative and nondeclarative memory

We tested the proposal that trace and delay eyeblink conditioning are fundamentally different kinds of learning. Strings of one, two, three, or four trials with the conditioned stimulus (CS) alone and strings of one, two, three, or four trials with paired presentations of both the CS and the unconditioned stimulus (US) occurred in such a way that the probability of a US was independent of string length. Before each trial, participants predicted the likelihood of the US on the next trial. During both delay ( n =20) and trace ( n = 18) conditioning, participants exhibited high expectation of the US following strings of CS-alone trials and low expectation of the US following strings of CS-US trials a phenomenon known as the gambler's fallacy. During delay conditioning, conditioned responses (CRs) were not influenced by expectancy but by the associative strength of the CS and US. Thus, CR probability was high following a string of CS-US trials and low following a string of CS-alone trials. The results for trace conditioning were opposite. CR probability was high when expectancy of the US was high and low when expectancy of the US was low. The results show that trace and delay eyeblink conditioning are fundamentally different phenomena. We consider how the findings can be understood in terms of the declarative and nondeclarative memory systems that support eyeblink classical conditioning.     

The influence of temporal factors on automatic priming and conscious expectancy in a simple reaction time task

In a previous study, we reported a dissociation between subjective expectancy and motor behaviour in a simple associative learning task (Perruchet, Cleeremans, & Destrebecqz, 2006). According to previous conditioning studies (Clark, Manns, & Squire, 2001), this dissociation is observed when the to-be-associated events coterminate and thus overlap in time (a training regimen called delay conditioning), but not when they are separated by a temporal delay (trace conditioning). In this latter situation indeed, there tends to be a direct relationship between subjective expectancy and behaviour. In this study, we further investigated this issue in a series of experiments where conscious and unconscious components of performance were pitted against each other. In Experiments 1–3, participants performed a simple reaction time task in which a preparatory signal (a tone) either overlapped with or terminated earlier than the imperative stimulus (a visual target presented in 50% of the trials). After each response, participants also had to state how much they expected the imperative stimulus to be displayed on the next trial. Results indicate that reaction times tend to decrease when the tone is consistently followed by the visual target across successive trials, whereas conscious expectancy for the target decreases at the same time. Importantly, we systematically found that the temporal relationship between the tone and the target failed to influence performance. In a fourth experiment, we examined whether these results extend to a two-choice reaction time task. To our surprise, we observed a direct relationship between subjective expectancies and reaction time in that situation. We nevertheless observed that the introduction of a delay between the tone and the target had, once again, no effect on performance.     

Dissociating expectancy of shock and changes in skin conductance: an investigation of the Perruchet effect using an electrodermal paradigm

Is human Pavlovian conditioning driven by a unitary, propositional system (as claimed by Mitchell, De Houwer, & Lovibond, 2009) or by dual systems; one under conscious control, symbolic in nature, and requiring effort to deploy, and the other utilizing associative processes and automatic in its operation (McLaren, Green, & Mackintosh, 1994)? Past research has suggested that for electrodermal conditioning to occur in humans, conscious awareness of the contingencies is necessary to produce conditioned responding (e.g., Hinchy, Lovibond, & Ter-Horst, 1995), as predicted by single process theories that attribute the conditioned response (CR) to conscious expectancy of the shock. In this article, the authors examined the Perruchet effect (Perruchet, 1985), using an electrodermal paradigm to determine whether there is any role for associative processes in human electrodermal conditioning. The authors attempted to replicate the basic effect, whereby expectancy of an unconditioned stimulus (US) increases over a run of nonreinforced trials while the CR to the conditional stimulus (CS) declines, and the complementary pattern in which expectancy decreases over a run of reinforced trials while the CR to the CS grows in strength. In line with these patterns, the change in skin conductance response (our CR) as a function of US run length was found to follow a linear trend opposite to that of conscious expectancy of shock with respect to US run length. This dissociation supports a dual-processing system account of human Pavlovian conditioning, with conscious, controlled processes governing expectancy (and subject to the gambler's fallacy), whereas automatic, associative processes determine at least some of the strength of the CR to the CS.     

Effects of Early Hippocampal Lesions on Trace, Delay, and Long-Delay Eyeblink Conditioning in Developing Rats

The effects of bilateral hippocampal aspiration lesions on later acquisition of eyeblink conditioning were examined in developing Long-Evans rat pups. Lesions on postnatal day (PND) 10 were followed by evaluation of trace eyeblink conditioning (Experiment 1) and delay eyeblink conditioning (Experiment 2) on PND 25. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US, 100 ms) were presented in one of three conditioning paradigms: trace (380 ms CS, 500 ms trace interval, 880 ms interstimulus interval [ISI]), standard delay (380 ms CS, 280 ms ISI), or long delay (980 ms CS, 880 ms ISI). The results of two experiments indicated that hippocampal lesions impaired trace eyeblink conditioning more than either type of delay conditioning. In light of our previous work on the ontogeny of trace, delay, and long-delay eyeblink conditioning (Ivkovich, Paczkowski, & Stanton, 2000) showing that trace and long-delay eyeblink conditioning had similar ontogenetic profiles, the current data suggest that during ontogeny hippocampal maturation may be more important for the short-term memory component than for the long-ISI component of trace eyeblink conditioning. The late development of conditioning over long ISIs may depend on a separate process such as protracted development of cerebellar cortex.     

Standard delay eyeblink classical conditioning is independent of awareness

P. F. Lovibond and D. R. Shanks (2002) suggested that all forms of classical conditioning depend on awareness of the stimulus contingencies. This article considers the available data for eyeblink classical conditioning, including data from 2 studies (R. E. Clark, J. R. Manns, & L. R. Squire, 2001; J. R. Manns, R. E. Clark, & L. R. Squire, 2001) that were completed too recently to have been considered in their review. In addition, in response to questions raised by P. F. Lovibond and D. R. Shanks, 2 new analyses of data are presented from studies published previously. The available data from humans and experimental animals provide strong evidence that delay eyeblink classical conditioning (but not trace eyeblink classical conditioning) can be acquired and retained independently of the forebrain and independently of awareness. This conclusion applies to standard conditioning paradigms; for example, to single-cue delay conditioning when a tone is used as the conditioned stimulus (CS) and to differential delay conditioning when the positive and negative conditioned stimuli (CS+ and CS-) are a tone and white noise.     

Parallel acquisition of awareness and differential delay eyeblink conditioning

There is considerable debate about whether differential delay eyeblink conditioning can be acquired without awareness of the stimulus contingencies. Previous investigations of the relationship between differential-delay eyeblink conditioning and awareness of the stimulus contingencies have assessed awareness after the conditioning session was finished using a post-experimental questionnaire. In two experiments, the point at which contingency awareness developed during the conditioning session was estimated from a button-press measure of expectancy of the unconditioned stimulus (US). In both experiments, knowledge of the stimulus contingencies and acquisition of differential delay eyeblink conditioning developed approximately in parallel. In Experiment 1 it was shown that predicting the US facilitated eyeblink conditioning compared with predicting the eyeblink response. In Experiment 2, a masking task was used that slowed down the emergence of awareness, and it was shown that differential conditioning only occurred in participants who were able to predict the US. The current findings challenge the hypothesis that differential delay eyeblink conditioning is entirely mediated by a functionally and neurally distinct nondeclarative learning system.     

Metabolic mapping of the rat cerebellum during delay and trace eyeblink conditioning

The essential neural circuitry for delay eyeblink conditioning has been largely identified, whereas much of the neural circuitry for trace conditioning has not been identified. The major difference between delay and trace conditioning is a time gap between the presentation of the conditioned stimulus (CS) and the unconditioned stimulus (US) during trace conditioning. It is this time gap or trace interval which accounts for an additional memory component in trace conditioning. Additional neural structures are also necessary for trace conditioning, including hippocampus and prefrontal cortex. This addition of forebrain structures necessary for trace but not delay conditioning suggests other brain areas become involved when a memory gap is added to the conditioning parameters. A metabolic marker of energy use, radioactively labeled glucose analog, was used to compare differences in glucose analog uptake between delay, trace, and unpaired experimental groups in order to identify new areas of involvement within the cerebellum. Known structures such as the interpositus nucleus and lobule HVI showed increased activation for both delay and trace conditioning compared to unpaired conditioning. However, there was a differential amount of activation between anterior and posterior portions of the interpositus nucleus between delay and trace, respectively. Cerebellar cortical areas including lobules IV and V of anterior lobe, Crus I, Crus II, and paramedian lobule also showed increases in activity for delay conditioning but not for trace conditioning. Delay and trace eyeblink conditioning both resulted in increased metabolic activity within the cerebellum but delay conditioning resulted in more widespread cerebellar cortical activation.     

Parallel acquisition of awareness and trace eyeblink classical conditioning

Trace eyeblink conditioning (with a trace interval ≥500 msec) depends on the integrity of the hippocampus and requires that participants develop awareness of the stimulus contingencies (i.e., awareness that the conditioned stimulus [CS] predicts the unconditioned stimulus [US]). Previous investigations of the relationship between trace eyeblink conditioning and awareness of the stimulus contingencies have manipulated awareness or have assessed awareness at fixed intervals during and after the conditioning session. In this study, we tracked the development of knowledge about the stimulus contingencies trial by trial by asking participants to try to predict either the onset of the US or the onset of their eyeblinks during differential trace eyeblink conditioning. Asking participants to predict their eyeblinks inhibited both the acquisition of awareness and eyeblink conditioning. In contrast, asking participants to predict the onset of the US promoted awareness and facilitated conditioning. Acquisition of knowledge about the stimulus contingencies and acquisition of differential trace eyeblink conditioning developed approximately in parallel (i.e., concurrently).     

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.     

Unimpaired trace classical eyeblink conditioning in Purkinje cell degeneration (pcd) mutant mice

Young adult Purkinje cell degeneration (pcd) mutant mice, with complete loss of cerebellar cortical Purkinje cells, are impaired in delay eyeblink classical conditioning. In the delay paradigm, the conditioned stimulus (CS) overlaps and coterminates with the unconditioned stimulus (US), and the cerebellar cortex supports normal acquisition. The ability of pcd mutant mice to acquire trace eyeblink conditioning in which the CS and US do not overlap has not been explored. Recent evidence suggests that cerebellar cortex may not be necessary for trace eyeblink classical conditioning. Using a 500 ms trace paradigm for which forebrain structures are essential in mice, we assessed the performance of homozygous male pcd mutant mice and their littermates in acquisition and extinction. In contrast to results with delay conditioning, acquisition of trace conditioning was unimpaired in pcd mutant mice. Extinction to the CS alone did not differ between pcd and littermate control mice, and timing of the conditioned response was not altered by the absence of Purkinje cells during acquisition or extinction. The ability of pcd mutant mice to acquire and extinguish trace eyeblink conditioning at levels comparable to controls suggests that the cerebellar cortex is not a critical component of the neural circuitry underlying trace conditioning. Results indicate that the essential neural circuitry for trace eyeblink conditioning involves connectivity that bypasses cerebellar cortex.     

A pitfall for the expectancy theory of human eyelid conditioning

Two simple eyeblink conditioning experiments with random intermittent reinforcement schedules were performed. In Experiment 1, subjects had to rate their expectancy for an unconditioned stimulus (US) on a seven-level scale prior to each trial. As anticipated, expectancy for US increased with a successive conditioned stimulus (CS) alone, and decreased with successive CS-US pairings. However, Experiments 1 and 2 showed that the frequency of eyeblink conditioned responses (CRs) evolved in a direction opposite to that of expectancy changes: CRs increased, whereas expectancy for US decreased, and vice versa. The possible effect of sensitization on eyeblink response was ruled out by the lack of a run effect in an unpaired control group in Experiment 2. These results tend to disconfirm the expectancy theory of conditioning. Although they were explicitly predicted by the conventional “strength” theory of conditioning, an alternative interpretation is proposed within a cognitive framework.     

Classical conditioning,awareness, and brain systems

Memory is composed of several different abilities that are supported by different brain systems. The distinction between declarative (conscious) and nondeclarative (non-conscious) memory has proved useful in understanding the nature of eyeblink classical conditioning – the best understood example of classical conditioning in vertebrates. In delay conditioning, the standard procedure, conditioning depends on the cerebellum and brainstem and is intact in amnesia. Trace conditioning, a variant of the standard procedure, depends additionally on the hippocampus and neocortex and is impaired in amnesia. Recent studies have sharpened the contrast between delay and trace conditioning by exploring the importance of awareness. We discuss these new findings in relation to the brain systems supporting eyeblink conditioning and suggest why awareness is important for trace conditioning but not for delay conditioning.     

Single-cue delay eyeblink conditioning is unrelated to awareness

We examined the importance of awareness for eyeblink conditioning by directly comparing singlecue delay eyeblink conditioning and single-cue trace eyeblink conditioning. During single-cue delay conditioning, participants who became aware of the stimulus contingencies early in the conditioning session conditioned no better than those who became aware later in the session or did not become aware. Thus, the level of awareness was unrelated to the overall level of conditioning across the session. In contrast, awareness of the stimulus contingencies early in the session predicted the success of single-cue trace conditioning. These data, together with earlier findings, show that awareness is irrelevant to single-cue delay eyeblink conditioning but is critical for single-cue trace eyeblink conditioning. The findings from the present study are related to previous findings for differential (CS⁺ and CS⁻) eyeblink conditioning and awareness.     

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.     

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.     

Eyeblink conditioning and systems consolidation: an ironic yet powerful pairing

Systems consolidation involves a prolonged process of memory reorganization that appears to be distinctly related to declarative memory. Declarative memory can be sharply contrasted with simple delay eyeblink classical conditioning, a prototypical example of nondeclarative memory. Yet inserting a trace interval between the conditioned and unconditioned stimuli endows eyeblink (trace) conditioning with many features of declarative memory. Work in humans has established that trace conditioning requires declarative memory. Recently trace eyeblink conditioning in animals has become one of the most powerful methods to study systems consolidation. Thus, it is ironic that a substantially nondeclarative form of memory has been so instructive concerning the organization of declarative memory.     

Cortical barrel lesions impair whisker-CS trace eyeblink conditioning

Whisker deflection is an effective conditioned stimulus (CS) for trace eyeblink conditioning that has been shown to induce a learning-specific expansion of whisker-related cortical barrels, suggesting that memory storage for an aspect of the trace association resides in barrel cortex. To examine the role of the barrel cortex in acquisition and retrieval of trace eyeblink associations, the barrel cortex was lesioned either prior to (acquisition group) or following (retention group) trace conditioning. The acquisition lesion group was unable to acquire the trace conditioned response, suggesting that the whisker barrel cortex is vital for learning trace eyeblink conditioning with whisker deflection as the CS. The retention lesion group exhibited a significant reduction in expression of the previously acquired conditioned response, suggesting that an aspect of the trace association may reside in barrel cortex. These results demonstrate that the barrel cortex is important for both acquisition and retention of whisker trace eyeblink conditioning. Furthermore, these results, along with prior anatomical whisker barrel analyses suggest that the barrel cortex is a site for long-term storage of whisker trace eyeblink associations.     

Timing of fear expression in trace and delay conditioning measured by fear-potentiated startle in rats

In two experiments, the time course of the expression of fear in trace (hippocampus-dependent) versus delay (hippocampus-independent) conditioning was characterized with a high degree of temporal specificity using fear-potentiated startle. In experiment 1, groups of rats were given delay fear conditioning or trace fear conditioning with a 3- or 12-sec trace interval between conditioned stimulus (CS) offset and unconditioned stimulus (US) onset. During test, the delay group showed fear-potentiated startle in the presence of the CS but not after its offset, whereas the trace groups showed fear-potentiated startle both during the CS and after its offset. Experiment 2 compared the time course of fear expression after trace conditioning with the time course in two delay conditioning groups: one matched to the trace conditioning group with respect to CS duration, and the other with respect to ISI. In all groups, fear was expressed until the scheduled occurrence of the US and returned to baseline rapidly thereafter. Thus, in both trace and delay fear conditioning, ISI is a critical determinant of the time course of fear expression. These results are informative as to the possible role of neural structures, such as the hippocampus, in memory processes related to temporal information.     

Learning in cod (Gadus morhua): long trace interval retention

Basic knowledge about learning capacities and awareness in fish is lacking. In this study we investigated which temporal gaps Atlantic cod could tolerate between two associated events, using an appetitive trace-conditioning paradigm with blinking light as conditioned stimulus (CS) and dry fish food as unconditioned stimulus (US). CS–US presentations were either temporally overlapping (delay conditioning, CS duration 24 s, interstimulus interval 12 s) or separated by 20, 60, or 120 s (trace conditioning, CS duration 12 s) or 2 h (control, CS duration 12 s). The percentage of fish in the feeding area increased strongly during CS presentation in all delay, 20 s, and 60 s trace groups and in one out of two 120 s trace groups, but not in the control groups. In the 20 and 60 s trace procedures, the fish crowded together in the small feeding area during the trace interval, showing strong anticipatory behaviour. In all the conditioned groups, the fish responded to the CS within eight trials, demonstrating rapid learning. At 88 and 70 days after the end of the conditioning experiments, the delay and 20 s trace groups, respectively, were presented the CS six times at 2-h intervals without reward. All groups responded to the light signal, demonstrating memory retention after at least 3 months. This study demonstrates that Atlantic cod has an impressively good ability to associate two time-separated events and long time retention of learnt associations.