A Renewed Interest in Human Classical Eyeblink Conditioning

Abstract—Over the past few years, a number of publications have reported the results of a variety of human classical eyeblink conditioning experiments. This renewed interest in human classical eyeblink conditioning appears to be caused by several factors, including the preference by some researchers to observe and report on behavior directly, the discovery that eyeblink conditioning can be used to assess basic biological and psychological processes, recent success in using eyeblink conditioning to determine the cause and expression of brain pathologies, and the successful use of this simple behavioral procedure in human imaging and electrophysiological experiments.     

New Directions for a Classical Paradigm: Human Eyeblink Conditioning

Abstract—The knowledge base on neural substrates an mechanisms involved in classical eyeblink conditioning makes it an ideal paradigm for investigating fundamental issues in learning and memory. New applications for the model system presented here include its use in (a) assessment to evaluate neurocognitive development in infancy, (b) theory building in abnormal psychology to test relationships between obsessive-compulsive behavior and learning rate, (c) evaluation of hypotheses about brain memory systems, and (d) exploration of the role of brain structures such as the cerebellum in learning and timing. Human eyeblink conditioning is a prototype of the utility of a model system that has become well characterized at both the behavioral and the neurobiological levels.     

Classical Eyeblink Conditioning: Clinical Models and Applications

In this paper, we argue that the main reason that classical eyeblink conditioning has proven so useful when applied to clinical situations, is that a great deal of information is known about the behavioral and neural correlates of this form of associative learning. Presented here is a summary of three lines of research that have used classical eyeblink conditioning to study three different clinical conditions; autism, fetal alcohol syndrome, and obsessive-compulsive disorder. While seemingly very different clinical conditions, classical eyeblink conditioning has proven very useful for advancing our understanding of these clinical pathologies and the neural conditions that may underlie them.     

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.     

Timing in Eyeblink Classical Conditioning and Timed-Interval Tapping

Abstract—The cerebellum is implicated in interval timing for diverse tasks including eyeblink classical conditioning (EBCC) and repetitive tapping. We examined performance on both tasks across identical intervals ranging from 325 to 550 ms. In five weekly sessions, 23 participants used a different interval each week, both as the target for tapping and as the delay interval in EBCC. Changes in variability as a function of the tapping or delay interval were assessed using regression analyses. The slope for repetitive tapping was comparable to two measures of temporal acuity in EBCC, onset and peak latency of the conditioned response. Each of 80 additional participants was assessed in one session at one of four tapping and delay intervals. Results were similar to those observed in the repeated measures group. These findings provide further evidence that EBCC and repetitive tapping utilize common mechanisms for representing temporal information.     

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.     

Eyeblink Classical Conditioning Differentiates Normal Aging from Alzheimer's Disease

Eyeblink classical conditioning is a useful paradigm for the study of the neurobiology of learning, memory, and aging, which also has application in the differential diagnosis of neurodegenerative diseases expressed in advancing age. Converging evidence from studies of eyeblink conditioning in neurological patients and brain imaging in normal adults document parallels in the neural substrates of this form of associative learning in humans and non-human mammals. Age differences in the short-delay procedure (400 ms CS-US interval) appear in middle age in humans and may be caused at least in part by cerebellar cortical changes such as loss of Purkinje cells. Whereas the hippocampus is not essential for conditioning in the delay procedure, disruption of hippocampal cholinergic neurotransmission impairs acquisition and slows the rate of learning. Alzheimer's disease (AD) profoundly disrupts the hippocampaL cholinergic system, and patients with AD consistently perform poorly in eyeblink conditioning. We hypothesize that disruption of hippocampal cholinergic pathways in AD in addition to age-associated Purkinje cell loss results in severely impaired eyeblink conditioning. The earliest pathology in AD occurs in entorhinal cortical input to hippocampus, and eyeblink conditioning may detect this early disruption before declarative learning and memory circuits become impaired. A case study is presented in which eyeblink conditioning detected impending dementia six years before changes on other screening tests indicated impairment. Because eyeblink conditioning is simple, non-threatening, and non-invasive, it may become a useful addition to test batteries designed to differentiate normal aging from mild cognitive impairment that progresses to AD and AD from other types of dementia.     

Eyeblink Conditioning in Rats Using Pontine Stimulation as a Conditioned Stimulus

Previous studies using rabbits and ferrets found that electrical stimulation of the pontine nuclei or middle cerebellar peduncle could serve as a conditioned stimulus (CS) in eyeblink conditioning (Bao, Chen, & Thompson, 2000; Hesslow, Svensson, & Ivarsson, 1999; Steinmetz, 1990; Steinmetz, Lavond, & Thompson, 1985; 1989; Steinmetz et al., 1986; Tracy, Thompson, Krupa, & Thompson, 1998). The current study used electrical stimulation of the pontine nuclei as a CS to establish eyeblink conditioning in rats. The goals of this study were to develop a method for directly activating the CS pathway in rodents and to compare the neural circuitry underlying eyeblink conditioning in different mammalian species. Rats were given electrical stimulation through a bipolar electrode implanted in the pontine nuclei paired with a periorbital shock unconditioned stimulus (US). Paired training was followed by extinction training. A subset of rats was given a test session of paired training after receiving an infusion of muscimol into the anterior interpositus nucleus. Rats given paired presentations of the stimulation CS and US developed CRs rapidly and showed extinction. Muscimol infusion prior to the test session resulted in a reversible loss of the eyeblink CR. The results demonstrate that electrical stimulation of the pontine nuclei can be used as a CS in rodents and that the CS pathway is similar in rats, rabbits, and ferrets. In addition, the loss of CRs following muscimol inactivation shows that the conditioning produced with pontine stimulation depends on cerebellar mechanisms.     

Effects of Emotional Valence and Arousal Manipulation on Eyeblink Classical Conditioning and Autonomic Measures

Using a classical eyeblink conditioning paradigm, we have previously shown that the rate of acquisition of a conditioned response may be manipulated by engaging subjects in background tasks of varying complexity concurrent to conditioning. To further examine the influence of the background environment on conditioning, a picture set designed to elicit emotional responses, the International Affective Picture System (IAPS), was presented to subjects during classical eyeblink conditioning. The results suggest that eyeblink conditioning does appear to be sensitive to contextual manipulations of arousal. Pictures rated as very arousing were found to engage subjects enough to enhance learning, although not to the point that autonomic functions were significantly altered between picture groups. We suggest that group differences in learning may be a result of either novelty of, or vigilance to, interesting pictures rather than as a direct result of physiological arousal.     

A Comparison of Latent Inhibition and Learned Irrelevance Pre-Exposure Effects in Rabbit and Human Eyeblink Conditioning

The learning of an association between a CS and a US can be retarded by unreinforced presentations of the CS alone (termed latent inhibition or LI) or by un-correlated presentations of the CS and US (termed learned irrelevance or LIRR). In rabbit eyeblink conditioning, there have been some recent failures to replicate LI. LIRR has been hypothesized as producing a stronger retardation effect than LI based on both empirical studies and computational models. In the work presented here, we examined the relative strength of LI and LIRR in eyeblink conditioning in rabbits and humans. In both species, a number of preexposure trials sufficient to produce LIRR failed to produce LI (Experiments 1 & 3). Doubling the number of CS pre-exposures did produce LI in rabbits (Experiment 2), but not in humans (Experiment 4). LI was demonstrated in humans only after manipulations including an increased inter-trial interval or ITI (Experiment 5). Overall, it appears that LIRR is a more easily producible pre-exposure retardation effect than LI for eyeblink conditioning in both rabbits and humans. Several theoretical mechanisms for LI including the conditioned attention theory, stimulus compression, novelty, and the switching theory are discussed as possible explanations for the differences between LIRR and LI. Overall, future work involving testing the neural substrates of pre-exposure effects may benefit from the use of LIRR rather than LI.     

The Effect of Scopolamine in Older Rabbits Tested in the 750 ms Delay Eyeblink Classical Conditioning Procedure

We investigated the effect of several doses of scopolamine in older rabbits that were trained for 20 days in the 750 ms delay eyeblink classical conditioning procedure. Our aim was to determine if the scopolamine-injected older rabbit would be a useful model for testing drugs for cognition enhancement in Alzheimer's disease (AD). A total of 39 rabbits with a mean age of 31 months received classical eyeblink conditioning with daily injections of 0.25, 0.75, or 1.5 mg/kg scopolamine hydrobromide or sterile saline vehicle. Doses of 0.75 and 1.5 mg/kg scopolamine significantly impaired acquisition, whereas acquisition was not significantly impaired with 0.25 mg/kg scopolamine. Results exhibit parallels in performance on delay eyeblink classical conditioning between scopolamine-treated older rabbits and human patients diagnosed with AD.     

Using Eyeblink Classical Conditioning as a Test of the Functional Consequences of Exposure of the Developing Cerebellum to Alcohol

Exposure of the developing brain to alcohol produces profound Purkinje cell loss in the cerebellum, and deficits in tests of motor coordination. However, the precise relationship between these two sets of findings has been difficult to determine. Eyeblink classical conditioning is known to engage a discrete brainstem-cerebellar circuit, making it an ideal test of cerebellar functional integrity after developmental alcohol exposure. In eyeblink conditioning, one of the deep cerebellar nuclei, the interpositus nucleus, as well as specific Purkinje cell populations, are sites of convergence for CS and US information. A series of studies have shown that eyeblink conditioning is impaired in both weanling and adult rats given binge-like exposure to alcohol as neonates, and that these deficits can be traced, at least in part, to impaired activation of cerebellar interpositus nucleus neurons and to an overall reduction in the deep cerebellar nuclear cell population. Because particular cerebellar cell populations are utilized in well-defined ways during eyeblink conditioning, conclusions regarding specific changes in the mediation of behavior by these cell populations are greatly strengthened. Further studies will be directed towards the impact of early exposure to alcohol on the functionality of specific Purkinje cell populations, as well as towards brainstem areas that process the tone CS and the somatosensory US.     

Ontogenetic Changes in the Neural Mechanisms of Eyeblink Conditioning

The rodent eyeblink conditioning paradigm is an ideal model system for examining the relationship between neural maturation and the ontogeny of associative learning. Elucidation of the neural mechanisms underlying the ontogeny of learning is tractable using eyeblink conditioning because the necessary neural circuitry (cerebellum and interconnected brainstem nuclei) underlying the acquisition and retention of the conditioned response (CR) has been identified in adult organisms. Moreover, the cerebellum exhibits substantial postnatal anatomical and physiological maturation in rats. The eyeblink CR emerges developmentally between postnatal day (PND) 17 and 24 in rats. A series of experiments found that the ontogenetic emergence of eyeblink conditioning is related to the development of associative learning and not related to changes in performance. More recent studies have examined the relationship between the development of eyeblink conditioning and the physiological maturation of the cerebellum, a brain structure that is necessary for eyeblink conditioning in adult organisms. Disrupting cerebellar development with lesions or antimitotic treatments impairs the ontogeny of eyeblink conditioning. Studies of the development of physiological processes within the cerebellum have revealed striking ontogenetic changes in stimulus-elicited and learning-related neuronal activity. Neurons in the interpositus nucleus and Purkinje cells in the cortex exhibit developmental increases in neuronal discharges following the unconditioned stimulus (US) and in neuronal discharges that model the amplitude and time-course of the eyeblink CR. The developmental changes in CR-related neuronal activity in the cerebellum suggest that the ontogeny of eyeblink conditioning depends on the development of mechanisms that establish cerebellar plasticity. Learning and the induction of neural plasticity depend on the magnitude of the US input to the cerebellum. The role of developmental changes in the efficacy of the US pathway has been investigated by monitoring neuronal activity in the inferior olive and with stimulation techniques. The results of these experiments indicate that the development of the conditioned eyeblink response may depend on dynamic interactions between multiple developmental processes within the eyeblink neural circuitry.     

Comparison of Single Unit Responses to Tone, Light, and Compound Conditioned Stimuli during Rabbit Classical Eyeblink Conditioning

Unit recordings and lesion studies have implicated the cerebellum as an essential site for the acquisition and maintenance of the conditioned eyeblink response. The current study looked at the neural characteristics of conditioned stimulus (CS) processing in the interpositus nucleus of the cerebellum after training New Zealand white rabbits (Oryctolagus cuniculus) in one of two conditioning paradigms: (a) compound conditioning (CMP), a compound CS consisting of light and tone paired with an air puff unconditioned stimulus (US); or (b) stimulus compounding (ALT), alternating blocks of tone CS and light CS trials paired with the air puff US. Single unit responses were recorded during five sessions after the animals had reached an asymptotic level of responding. Animals were tested for behavioral and neural responses to CS alone trials that included tone alone, light alone, and compound tone-light trials. For the CMP group, the compound CS elicited 80 to 90% conditioned eyeblink responses (CRs), whereas the individual tone and light CSs elicited only 40 to 50% CRs. For the ALT group, all three CSs (tone, light, and compound) elicited very high levels of responding of at least 80% CRs. For the CMP group, there were roughly equal numbers of cells responding to all of the CSs. This includes cells that responded exclusively to one, and only one, of the three stimuli and also those cells that responded to combinations of two or more. Cells from the ALT group were far more likely to respond exclusively to only one of the CSs. Both the behavioral and physiological results suggest that the compound tone-light stimulus was processed as a distinct stimulus, separate from the component tone and light. These results are discussed in the context of multisensory processing.     

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.     

Effects of US-Alone Presentations on Human Evaluative Conditioning

In the context of evaluative conditioning, the effects of additional presentations of the unconditioned stimulus (US) prior to conditioning (US preexposure) or after conditioning (US postexposure) were examined using between- and within-subjects control conditions. Two experiments that differed with respect to the nationality of the subjects were conducted. In both experiments, US-alone presentations reduced the magnitude of the evaluative response. The US pre- and postexposure effects were observed in subjects classified as aware as well as in subjects classified as unaware of the experimental contingencies. Another finding is that the evaluative conditioning procedure described by Martin and Levey (1978; Levey & Martin, 1975) resulted in reliable conditioning effects also in an American sample, thus extending the scope of that special evaluative conditioning paradigm. The findings are discussed in the context of recent models of classical and evaluative conditioning. Especially, the unexpected US postexposure effect gives rise to speculations concerning the learning process underlying evaluative conditioning.     

Discrimination Reversal Conditioning of an Eyeblink Response is Impaired by NMDA Receptor Blockade

In the present study we examined the effects of the specific NMDA receptor antagonist CPP on discrimination reversal learning in rabbits. We report two primary findings. First, the institution of NMDA receptor blockade had no effect on a learned discrimination. Second, after stimulus reversal, CPP treatment impaired acquisition of the discrimination reversal. This impairment manifested itself early in training as a retardation in acquisition of a CR to the new CS+ and late in training as an inability to suppress responsiveness to the new CS-. Given the comparability of the present results with previously published results for phenytoin-treated rabbits, we suggest that the effects of phenytoin on learning in this paradigm is at least in part mediated by its effects on NMDA receptors. We further suggest that these findings emphasize the need to better define the role of NMDA receptor activation and hippocampally-mediated circuits in a variety of associative learning paradigms.