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.     

Age-related impairment in the 250-millisecond delay eyeblink classical conditioning procedure in C57BL/6 mice

In this study we tested 4-, 9-, 12-, and 18-month-old C57BL/6 mice in the 250-msec delay eyeblink classical conditioning procedure to study age-related changes in a form of associative learning. The short life expectancy of mice, complete knowledge about the mouse genome, and the availability of transgenic and knock-out mouse models of age-related impairments make the mouse an excellent species for expanding knowledge on the neurobiologically and behaviorally well-characterized eyeblink classical conditioning paradigm. Based on previous research with delay eyeblink conditioning in rabbits and humans, we predicted that mice would be impaired on this cerebellar-dependent associative learning task in middle-age, at ~9 months. To fully examine age differences in behavior in mice, we used a battery of additional behavioral measures with which to compare young and older mice. These behaviors included the acoustic startle response, prepulse inhibition, rotorod, and the Morris water maze. Mice began to show impairment in cerebellar-dependent tasks such as rotorod and eyeblink conditioning at 9 to 12 months of age. Performance in hippocampally dependent tasks was not impaired in any group, including 18-month-old mice. These results in mice support results in other species, indicating that cerebellar-dependent tasks show age-related deficits earlier in adulthood than do hippocampally dependent tasks.     

Neurotransmitter release during delay eyeblink classical conditioning: role of norepinephrine in consolidation and effect of age

Delay classical eyeblink conditioning (EBC) is an important model of associative, cerebellar-dependent learning. Norepinephrine (NE) plays a significant modulatory role in the acquisition of learning; however, other neurotransmitters are also involved. The goal was to determine whether NE, gamma-aminobutyric acid (GABA) and glutamate (GLU) release are observed in cerebellar cortex during EBC, and whether such release was selectively associated with training. Further studies examined the role of the beta-noradrenergic receptor in consolidation of the learned response by local infusion of propranolol at 5-120 min following training into the cerebellar cortex. In vivo microdialysis coupled to EBC was performed to examine neurotransmitter release. An increase in the extracellular level of NE was observed during EBC and was maximal on day 1 and diminished in amplitude with subsequent days of training. No changes in baseline NE release were observed in pseudoconditioning indicating that NE release is directly related to the associative learning process. The extracellular levels of GABA were also increased selectively during paired training however, the magnitude of GABA release increased over days of training. GLU release was observed to increase during both paired and unpaired training, suggesting that learning does not occur prior to the information arriving in the cerebellum. When propranolol was administered at either 5-, 60-, or 120-min post-training, there was an inhibition of conditioned responses, these data support the hypothesis that NE is important for consolidation of learning. In another set of experiments we demonstrate that the timing of release of NE, GABA and glutamate are significantly delayed in onset and lengthened in duration in the 22-month-old F344 rats. Over days of training the timing of release becomes closer to the timing of training and this is associated with increased learning of conditioned responses in the aged rats.     

Effects of paradigm and inter-stimulus interval on age differences in eyeblink classical conditioning in rabbits

The aim of this study was to examine parameters affecting age differences in eyeblink classical conditioning in a large sample of young and middle-aged rabbits. A total of 122 rabbits of mean ages of 4 or 26 mo were tested at inter-stimulus intervals (ISIs) of 600 or 750 msec in the delay or trace paradigms. Paradigm affected both age groups dramatically, with superior performance in the delay paradigm. ISI was salient as middle-aged rabbits were significantly impaired in 750-msec compared with 600-msec delays, and young rabbits were significantly less impaired in 600-msec than in 750-msec trace. Young rabbits performed equally well at both delay ISIs, and consequently, there were significant age differences in 750-msec but not in 600-msec delays. Middle-aged rabbits performed poorly at both 600- and 750-msec trace, resulting in significant age differences in 600-msec but not in 750-msec trace. Timing of the conditioned response has been associated with cerebellar cortical function. Normal aging of the cerebellar cortex likely contributed to the magnitude of the effect of ISI in delay conditioning in middle-aged rabbits. Results demonstrate that the magnitude of age differences in eyeblink conditioning can be enlarged or eliminated by ISI and paradigm.     

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.     

Cerebellar norepinephrine modulates learning of delay classical eyeblink conditioning: evidence for post-synaptic signaling via PKA

The neurotransmitter norepinephrine (NE) has been shown to modulate cerebellar-dependent learning and memory. Lesions of the nucleus locus coeruleus or systemic blockade of noradrenergic receptors has been shown to delay the acquisition of several cerebellar-dependent learning tasks. To date, no studies have shown a direct involvement of cerebellar noradrenergic activity nor localized the post-synaptic response to cerebellar beta-noradrenergic receptor signaling. Using ipsilateral, localized infusions into cerebellar lobule HVI and interpositus (IP), we have established that blocking beta-noradrenergic receptors with propranolol significantly impairs acquisition of conditioned responses. Furthermore, interrupting activation of cAMP-dependent PKA in the cerebellum using Rp-cAMPS completely prevents acquisition. However, neither blocking beta-adrenergic receptors nor blocking PKA activation significantly interferes with performance of established conditioned responses when administered after the learned response is formed.     

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).     

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.     

Reevaluating the role of the medial prefrontal cortex in delay eyeblink conditioning

It has been proposed that the medial prefrontal cortex (mPFC) is not necessary for delay eyeblink conditioning (DEC). Here, we investigated the involvement of the mPFC in DEC with a soft or loud tone as the conditioned stimulus (CS) by using electrolytic lesions or muscimol inactivation of guinea pig mPFC. Interestingly, when a soft tone was used as a CS, electrolytic lesions of the mPFC significantly retarded acquisition of the conditioned response (CR), and muscimol infusions into mPFC distinctly inhibited the acquisition and expression of CR, but had no significant effect on consolidation of well-learned CR. In contrast, both electrolytic lesions and muscimol inactivation of mPFC produced no significant deficits in the CR when a loud tone was used as the CS, or in the unconditioned response (UR) when a soft or loud tone was used as the CS. These results demonstrate that the mPFC is essential for the DEC with the soft tone CS but not for the DEC with the loud tone CS.     

Stress impairs acquisition of delay eyeblink conditioning in men and women

In rodents stress impairs delay as well as trace eyelid conditioning in females, but enhances it in males. The present study tested the effects of acute psychosocial stress exposure on classical delay eyeblink conditioning in healthy men and women. In a between subject design, participants were exposed to psychosocial stress using the Trier Social Stress Test (TSST) or a control condition which was followed by a delay eyeblink classical conditioning procedure. Stress exposure led to a significant increase in salivary cortisol and impaired acquisition of conditioned eyeblink responses (CRs). This was evident by a later first CR and an overall lower CR rate of the stress group. The stress-induced acquisition impairment was observed in both women and men. Subjects failing to show a stress-induced cortisol increase (cortisol non-responder) were not impaired in acquisition. Our findings indicate that acute stress, possibly via activation of the hypothalamus–pituitary–adrenal (HPA) axis, reduces the ability to acquire a simple conditioned motor response in humans.     

Galantamine facilitates acquisition of hippocampus-dependent trace eyeblink conditioning in aged rabbits

Cholinergic systems are critical to the neural mechanisms mediating learning. Reduced nicotinic cholinergic receptor (nAChR) binding is a hallmark of normal aging. These reductions are markedly more severe in some dementias, such as Alzheimer's disease. Pharmacological central nervous system therapies are a means to ameliorate the cognitive deficits associated with normal aging and aging-related dementias. Trace eyeblink conditioning (EBC), a hippocampus- and forebrain-dependent learning paradigm, is impaired in both aged rabbits and aged humans, attributable in part to cholinergic dysfunction. In the present study, we examined the effects of galantamine (3 mg/kg), a cholinesterase inhibitor and nAChR allosteric potentiating ligand, on the acquisition of trace EBC in aged (30–33 months) and young (2–3 months) female rabbits. Trace EBC involves the association of a conditioned stimulus (CS) with an unconditioned stimulus (US), separated by a stimulus-free trace interval. Repeated CS–US pairings results in the development of the conditioned eyeblink response (CR) prior to US onset. Aged rabbits receiving daily injections of galantamine (Aged/Gal) exhibited significant improvements compared with age-matched controls in trials to eight CRs in 10 trial block criterion (P = 0.0402) as well as performance across 20 d of training [F(1,21) = 5.114, P = 0.0345]. Mean onset and peak latency of CRs exhibited by Aged/Gal rabbits also differed significantly [F(1,21) = 6.120/6.582, P = 0.0220/0.0180, respectively] compared with age-matched controls, resembling more closely CR timing of young drug and control rabbits. Galantamine did not improve acquisition rates in young rabbits compared with age-matched controls. These data indicate that by enhancing nicotinic and muscarinic transmission, galantamine is effective in offsetting the learning deficits associated with decreased cholinergic transmission in the aging brain.     

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.     

Contextual specificity of extinction of delay but not trace eyeblink conditioning in humans

Renewal of an extinguished conditioned response has been demonstrated in humans and in animals using various types of procedures, except renewal of motor learning such as eyeblink conditioning. We tested renewal of delay and trace eyeblink conditioning in a virtual environment in an ABA design. Following acquisition in one context (A, e.g., an airport) and extinction in a different context (B, e.g., a city), tests for renewal took place in the acquisition (A) and extinction context (B), in a counterbalanced order. Results showed renewal of the extinguished conditioned response in the delay but not trace condition.     

Hippocampal theta-dependent eyeblink classical conditioning: coordination of a distributed learning system

Richard F. Thompson's study of the neurobiological substrates of learning and memory has been a career-long endeavor, chosen early and pursued with uncompromising depth and breadth. His systematic mapping of the major brain systems and mechanisms involved in eyeblink classical conditioning (EBCC) established the essential role of the cerebellum. Investigations of the interactions between the hippocampus and cerebellum are critically important to this literature, given the essential involvement of these structures in trace EBCC as well as an important modulatory role of the hippocampus in delay EBCC. Hippocampal theta (3-7Hz) oscillations are known to reflect a functional state that influences both the timing of unit firing and the potential for neural plasticity in the hippocampus and other structures. Herein we present a brief summary of research demonstrating the behavioral enhancement due to theta and the underlying neurobiological correlates in both hippocampus and cerebellum during EBCC. Hippocampal and cerebellar local field potentials (LFPs) show that these distantly interconnected brain structures become precisely synchronized when conditions favor rapid behavioral acquisition. Our results suggest a major role for theta in coordinating the widely distributed memory system for trace EBCC. These and other important findings reflect Thompson's own work and his early-career mentoring of scientists whose contributions to the EBCC literature have ensured his major and lasting impact on the neurobiology of learning and memory.     

Differential effects of progesterone and medroxyprogesterone on delay eyeblink conditioning in ovariectomized rats

Ovarian hormones modulate acquisition processes involved in classical conditioning. Although progesterone has been indirectly implicated, its role in classical conditioning of the eyeblink response has not been directly investigated. We assessed the effects of daily dosing of progesterone or medroxyprogesterone (MPA), a non-metabolized synthetic progestin, upon the acquisition of a classically conditioned eyeblink response in ovariectomized (OVX) female rats. Rats were dosed 4h prior to each training session with 0.1 or 1.5 mg/kg of either of these hormones or sesame oil. A delay conditioning paradigm was employed using a 500 ms conditioned stimulus coterminating with a 10 ms 10 V unconditioned stimulus. At the low dose, progesterone and MPA rats did differ from each other, with MPA-treated rats learning slower, but neither group differed from OVX-oil or Sham-oil controls. No group differences in acquisition were observed at the higher dose. During extinction trials, high-dose MPA-treatment and OVX-oil groups extinguished quicker than the high-dose progesterone-treated group. In addition, unconditional response (UR) amplitudes were lower in all OVX groups, regardless of hormone or oil treatment, compared to the sham-oil group. Since MPA did not affect extinction, it is likely the slower extinction in the progesterone-treated rats is due to a metabolite of progesterone. Corticosterone is discussed as a likely candidate for such a role. In addition, we found chronic absence of ovarian hormones decreased UR amplitudes, although differences in UR amplitudes were not associated with changes in the acquisition process. These results are discussed with respect to differences in the hormonal effects upon acquisition versus extinction processes and how these data may explain reports of learning differences in women based on oral contraceptive usage.     

Retention and extinction of delay eyeblink conditioning are modulated by central cannabinoids

Rats administered the cannabinoid agonist WIN55,212-2 or the antagonist SR141716A exhibit marked deficits during acquisition of delay eyeblink conditioning, as noted by Steinmetz and Freeman in an earlier study. However, the effects of these drugs on retention and extinction of eyeblink conditioning have not been assessed. The present study examined the effects of WIN55,212-2 and SR141716A on retention and extinction of delay eyeblink conditioning in rats. Rats were given acquisition training for five daily sessions followed by one session of retention training with subcutaneous administration of 3 mg/kg of WIN55,212-2 or 5 mg/kg of SR141716A and an additional session with the vehicle. Two sessions of extinction training were then given with WIN55,212-2, SR141716A, or vehicle. Retention and extinction were impaired by WIN55,212-2, whereas SR141716A produced no deficits. The extinction deficit in rats given WIN55,212-2 was observed only during the first session, suggesting a specific impairment in short-term plasticity mechanisms. The current results and previous findings indicate that the cannabinoid system modulates cerebellar contributions to acquisition, retention, and extinction of eyeblink conditioning.     

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.     

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.