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).     

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.     

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.     

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.     

Blocking the BK channel impedes acquisition of trace eyeblink conditioning

Big-K⁺ conductance (BK)-channel mediated fast afterhyperpolarizations (AHPs) following action potentials are reduced after eyeblink conditioning. Blocking BK channels with paxilline increases evoked firing frequency in vitro and spontaneous pyramidal activity in vivo. To examine how increased excitability after BK-channel blockade affects learning, rats received bilateral infusions of paxilline, saline, or nothing into hippocampal CA1 prior to trace eyeblink conditioning. The drug group was slower to acquire the task, but learning was not completely impaired. This suggests that nonspecific increases in excitability and baseline neuronal firing rates caused by in vivo blockade of the BK channel may disrupt correct processing of inputs, thereby impairing hippocampus-dependent learning.Learning and increased neuronal intrinsic excitability are strongly correlated, although a causal relationship has not yet been definitively established (Disterhoft and Oh 2006). One of the mechanisms of increased excitability is through reduction of potassium currents, which cause afterhyperpolarizations (AHP). Afterhyperpolarizations in pyramidal cells can be divided into three categories based upon their timing and duration. The fast AHP lasts only 2–5 ms, follows the depolarizing phase of individual action potentials, and is mediated largely by the big-K⁺ conductance (BK) channel. The post-burst AHP has a medium (50–100 ms) and a slow (1–2 s) component, and follows trains or bursts of action potentials. The medium AHP is carried by apamin sensitive small-K⁺ conductance (SK) channels, but the channel(s), which carry the slow AHP, are still unknown (Storm 1987; Disterhoft and Oh 2006).Learning-related reductions in the post-burst AHP are well documented (for review see Disterhoft and Oh 2006). Additionally, pharmacological modulators of the post-burst AHP alter learning in an expected manner—compounds that reduce the AHP improve learning (galantamine [Simon et al. 2004] and nimodopine [Deyo et al. 1989]). Learning-related reductions of the fast AHP are also seen in prefrontal cortex pyramidal neurons after extinction of fear conditioning (Santini et al. 2008) and in CA1 hippocampal pyramidal neurons after learning trace eyeblink conditioning (tEBC) (Matthews et al. 2008). In vitro whole-cell recordings show that blocking the BK channel with either paxilline or iberiotoxin increases the firing rate to a step current injection (Nelson et al. 2003). Likewise, injection of paxilline into the hippocampus increases the in vivo spontaneous firing frequency of hippocampal CA1 neurons of awake freely moving rats up to 2.5-fold over saline injections (Matthews et al. 2008), indicating that the BK-mediated fast AHP plays an important role in intrinsic excitability. The current study was undertaken to determine whether pharmacologically reducing the fast AHP during training would improve trace eyeblink conditioning.Experimental subjects were 3- to 4-mo old Fisher 344 X Brown Norway F1-hybrid rats. Animals were housed in pairs, in a climate-controlled facility with a 12:12 light–dark cycle, and ad libitum access to food and water. Procedures were in accordance with the guidelines of the Northwestern University Animal Care and Use Committee and conformed to NIH standards (NIH Publication No. 80-23). All efforts were made to minimize the number of animals utilized and their discomfort. Thirty-seven rats were originally included in the study, however 13 were excluded from the experiment due to incorrect cannulae placement, faulty EMG signal, or unrelated health issues. The final groups included in the study were nine drug animals, seven vehicle animals, eight sham animals, and six non-cannulated animals.Guide cannulae (26 gauge stainless steel) were bilaterally implanted at −3.6 mm AP, ±2.0 mm ML. The guide cannulae were lowered slowly (0.5 mm/5 min) to a depth of −1.9 mm subdura. The cannulae were cemented in place with dental acrylic. An electronic connector strip with pins for ground and two EMG wires was fitted between the two guide cannulae and grounded to the skull screws. The EMG wires were implanted under the right eyelid and the entire apparatus was cemented in place (Weiss et al. 1999). Rats were given Buprenex (0.05 mg/kg) post-surgery to alleviate any discomfort.Cannulae placements were verified histologically after training was completed. Animals were given a lethal dose of barbiturate (0.15 mL i.p.) then transcardially perfused with 0.9% saline followed by 10% formalin. After perfusion, the brains were carefully immersed in 10% formalin for a minimum of 24 h. Eighty micron coronal sections were made with a freezing cryostat, and every second section was kept and mounted on gelatin coated slides. Sections were stained with cresyl violet to reveal cell death or excessive damage surrounding the injection cannulae. Animals with incorrectly placed cannulae or excessive tissue damage were not included in the study. A diagram of the most ventral extent of each cannula is shown in Figure 1A. Paxilline, a BK-channel blocker, is a peptide and there is little known about the motility of this molecule in the brain. From previous in vivo recording work (Matthews et al. 2008), it is known that the BK blocker is active and able to diffuse at least a radius of 1.7 mm from the tip of the cannula. To approximate the spread of paxilline in the present study, 1.0% ibotenic acid was injected into two animals at the completion of training in a manner that exactly mimicked the paxilline injections in volume and rate. Five days after excitotoxic lesion, animals were sacrificed and their brains processed. Figure 1B shows the maximum and minimum extent of cell loss due to ibotenic acid lesion.Open in a separate windowFigure 1.Cannula placement and drug spread. (A) Cannulae were bilaterally implanted to terminate directly above the CA1 layer of the hippocampus. Placement was verified after the behavioral experiments. Gray dots indicate the location of the tip of each cannula for animals included in the study. (B) The spread of the drug was approximated by injecting 1.0% ibotenic acid into the left hemisphere of two animals at the end of training. The injection volume and rate were the same as those used for the study. The right hemisphere served as a within-animal control for the action of the ibotenic acid. The maximal (light gray) and minimal (dark gray) spread are shown. Measurements are relative to bregma. (Adapted from Paxinos and Watson [1998] and reprinted with permission from Elsevier ©1998.)Freely moving animals were injected with vehicle (1% DMSO in saline), drug (1 μM paxilline in saline), or nothing 20–30 min before the start of training, including the first habituation session. The trainer was blind to the identity of the injected substance. Infusions were performed using two 2 μL Hamilton syringes connected by lengths of flexible, oil filled tubing to 33 gauge infusion needles, which extended 0.5 mm beyond the end of the guide cannulae. One microliter of sterile solution was infused into each hemisphere at a constant rate of 0.2 μL/min using a Stoelting double-barrel infusion pump. The injection needles were left in place for 1 min following the injection to allow diffusion away from the tip of the needle.Trace eyeblink conditioning is an associative learning task in which a neutral conditioned stimulus (CS) is paired with an unconditioned stimulus (US) that elicits a reflexive eyelid closure. The insertion of a stimulus-free “trace” interval between the CS and the US makes this task strongly dependent on the hippocampus (Solomon et al. 1986; Moyer Jr. et al. 1990). After repeated pairings of the CS and the US, if an association has been learned, the subject will begin to blink during the trace period in anticipation of the US. Trace eyeblink conditioning procedures as described by Weiss et al. (1999) were followed. Training sessions were conducted in a sound-attenuating chamber and controlled with a custom-designed LabVIEW (National Instruments) program; eyelid EMG data were integrated online during training. Animals were connected to the recording computer via the implanted connector strip; a short tether served the dual purpose of allowing EMG activity to be monitored and positioning an air puff delivery tube in front of the eye, while the rat was freely moving. On the first day, the subjects received a session of stimulus-free habituation to the training chamber lasting as long as a conditioning session. The subsequent 5 d were training sessions. Conditioned animals received 30 trials per session (30 s average ITI) for a total of 150 CS–US pairings, consisting of a tone stimulus (CS, 80 dB, 250 ms) paired with a corneal air puff (US, 3–5 psi, 100 ms) with a 250 ms stimulus-free trace interval interposed.The primary measure of tEBC learning used in this study was a correctly timed eyelid closure, i.e., an eyelid closure that begins during the trace interval and continues until the air puff. Eyelid activity was measured with an implanted EMG electrode. The division of eyelid responses into “adaptive” and “nonadaptive” categories has been used in other studies (Garcia et al. 1999). For this reason, we analyzed eyelid closure during the entire tone/trace period and during only the last 200 ms preceding the air puff. Responses given during the 200 ms preceding the US are termed adaptive conditioned responses (CR). Figure 2 shows the timing of the tone, trace, and air puff; the timeline for each type of response; and an integrated EMG. Any trials in which the baseline activity in the 500 ms preceding CS presentation exceeded two standard deviations were discarded. Eye closure was defined as greater than four standard deviations above baseline. Averages for all relevant measures for each session were compared between groups for training sessions 1–5 using repeated-measures ANOVA. Learning across training sessions within each group was assessed with a planned comparison ANOVA using StatVIEW software. The stimulus-free habituation session was excluded from all between-group analyses. The percentage of aCRs during habituation is shown in Figure 3 to provide the baseline level of spontaneous eyelid closures.Open in a separate windowFigure 2.Timing of conditioned responses (CRs) in relation to the tone, trace, and air puff. The integrated EMG shows a robust adaptive eyelid closure in anticipation of the air puff. The tone lasted 250 ms and was followed by a 250 ms trace period. The air puff was 100 ms long. Below the EMG trace is a time line showing the period for an adaptive CR and an unconditioned response (UR).Open in a separate windowFigure 3.BK block slows learning of trace eyeblink conditioning. Vehicle, drug, and sham animals were injected and immediately trained on the trace eyeblink task over 6 d, one session each day. (Non-cannulated animals were trained in two sessions per day for 3 d.) The first session for all groups was stimuli-free habituation. Each session consisted of 30 pairings of a CS tone with a US air puff. Injection of the BK blocker paxilline resulted in slower acquisition of the task (*P < 0.05), although animals in all groups were eventually able to learn the task (#P < 0.001).The concentration of paxilline was selected based on the dose that achieved a maximal increase in spontaneous firing in vivo (Matthews et al. 2008). Two groups were designed to control for pressure effects of the injection or the stress and tissue damage of the cannulation surgery. These were a vehicle group (1% DMSO in saline) and a sham group (empty needles). In addition, the learning behavior of these cannulated animals was compared to a separate group of animals that had only the EBC head apparatus implanted (non-cannulated).Rats that received an infusion of paxilline (1 μM) immediately preceding training were significantly slower to acquire the task as measured by the percent of adaptive CRs exhibited across all training sessions (F _(3,26) = 3.155, P = 0.042, repeated-measures ANOVA, Fisher''s PLSD, P < 0.02) (Fig. 3). The percentage of responses during the entire tone/trace period showed a trend toward reduced learning in the drug group (F _(3,26) = 2.729, P = 0.064, repeated-measures ANOVA).Other parameters of the eyelid closure were examined. There were no significant differences in the peak, onset, duration, or area of the adaptive CR. There was also no difference between groups in the onset of the air-puff elicited eyelid closure (unconditioned response [UR]), suggesting that the drug did not cause decreased sensitivity to the US. Finally, the baseline eyelid EMG activity of all four groups during the stimulus-free habituation session was not significantly different, suggesting that the drug did not suppress or enhance spontaneous eyelid activity.All animals in the study showed improved performance across the training sessions (F _(4,104) = 21.810, P < 0.0001, repeated-measures ANOVA). Further analyses revealed that animals in the drug group also showed continuous learning over the training days (Session 1: 16.1% ± 4.9%, Session 5: 56.8% ± 8.2%; F _(4,32) = 6.961; P = 0.0004), and eventually the drug group reached a percentage of adaptive CRs statistically comparable to the controls (F _(3,26) = 2.377, P = 0.093 for Session 5, ANOVA).There is a precedent for anticipating enhanced learning after in vivo pharmacological manipulations of intrinsic excitability (Disterhoft and Oh 2006). The long-lasting post-burst AHP is reduced in hippocampal cells from animals that have learned a hippocampus-dependent task (Moyer Jr. et al. 1996; Oh et al. 2003), and increased in aging animals that have difficulty learning. Pharmacologically reducing the post-burst AHP in vivo with calcium channel blockers (nimodipine) (Deyo et al. 1989), acetylcholine agonists (CI-1017) (Weiss et al. 2000), or cholinesterase inhibitors (galantamine and metrifonate) (Kronforst-Collins et al. 1997; Weible et al. 2004) leads to improved learning in aging animals. Thus, the finding that blocking the BK channel results in slowed learning is somewhat surprising, given the previous report of a reduction in the BK-mediated fast AHP after learning tEBC (Matthews et al. 2008). Several explanations may account for why pharmacologically reducing the fast AHP in vivo impaired rather than improved learning.First, the reduction of the fast AHP seen with channel blockers in in vitro experiments is of greater magnitude than the reductions in the fast AHP seen after learning; additionally, the fast AHP in cells from trained animals can be further reduced in vitro with paxilline or iberiotoxin (Matthews et al. 2008). It could be that there is an important difference between reducing the BK-mediated current, as is seen after learning, and completely blocking it using a drug. Reducing the fast AHP increases intrinsic excitability, and completely blocking the BK channel increases excitability even further. However, it is important to emphasize recent research showing how excessive excitability can be detrimental to learning. An early indicator of mild cognitive impairment detected with fMRI is hyperactivity in the hippocampus and medial temporal lobe (Miller et al. 2008). At the cellular level, saturating in vivo hippocampal LTP results in impaired spatial learning due to increased epileptiform activity, rather than saturated synaptic plasticity (McNamara et al. 1993). Finally, work with a knockout model of the β-4 subunit of the BK channel shows that this calcium- and voltage-dependent channel helps to regulate hyperexcitability and reduce the occurrence of temporal lobe seizures (Brenner et al. 2005). Although intrahippocampal paxilline infusion did not cause epileptiform activity in in vivo recordings, or observable seizure behavior (Juhng et al. 1999), it is possible that the drug caused pathological increases in excitability that impeded learning.Second, unregulated or meaningless increases in baseline neural activity in the hippocampus increase background noise, effectively decreasing the signal-to-noise ratio for the whole network, making it more difficult to distinguish important, information-bearing activity from background noise. In delay conditioning (where there is no trace interval between the CS and US), ablation of the hippocampus does not disrupt eyeblink conditioning (Solomon et al. 1986; Hesslow 1994), however, increasing (Salafia et al. 1979) or suppressing (Solomon et al. 1983) hippocampal activity has a strong retarding effect on learning this task. Disrupting synaptic transmission in a subregion of the hippocampus also impairs spatial learning (Niewoehner et al. 2007), further illustrating how a disturbance of information processing at a single junction of the trisynaptic circuit can impair learning. BK channels are present in presynaptic terminals, as well as at the soma, where they participate in controlling transmitter release. Blocking BK channels decreases failures and increases the amplitude of EPSCs at CA3–CA3 synapses (Raffaelli et al. 2004). The infusion of paxilline may have also increased the efficacy and frequency of transmitter release at the CA3–CA1 synapse in the present experiment. It may be that by selecting a dose of paxilline that caused a maximum increase in in vivo spontaneous activity, we overdosed the hippocampus to levels of excitability that interfered with stimulus processing, thereby impairing and slowing learning.Finally, the fast AHP is largely mediated by the BK channel, but other potassium currents also play a role in action potential repolarization. The A-type potassium current in particular has been implicated in learning-related excitability changes and is active during an action potential (Giese et al. 1998). Changing action potential repolarization dynamics also alters the calcium influx into cells (Zhou et al. 2005), which can have far reaching effects on other calcium-dependent processes, such as gene regulation, synaptic plasticity, or protein expression. The learning impairment seen in this study might be due to secondary effects on other potassium currents or calcium-dependent processes.This study indicates that normal activity of BK channels is required for acquisition of the tEBC task. The channel may act to maintain neurons within a narrow window of excitability, keeping neurons within an operating range of “optimal excitability.” BK channel activity is strongly modulated by kinase-phosphotase activity (Reinhart et al. 1991; Loane et al. 2006), and reduction of BK-mediated current through modulators of these molecules may have a more beneficial impact on learning. Since blocking of the BK channel with paxilline impedes learning, this drug may not present a useful tool for pharmacological learning-enhancement manipulations.     

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.     

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.     

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.     

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.     

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.     

Simultaneous training on two hippocampus-dependent tasks facilitates acquisition of trace eyeblink conditioning

A common cellular alteration, reduced post-burst afterhyperpolarization (AHP) in CA1 neurons, is associated with acquisition of the hippocampus-dependent tasks trace eyeblink conditioning and the Morris water maze. As a similar increase in excitability is correlated with these two learning paradigms, we sought to determine the interactive behavioral effects of training animals on both tasks by using either a consecutive or simultaneous training design. In the consecutive design, animals were trained first on either the trace eyeblink conditioning task for six sessions, followed by training on the water maze task for six sessions, or vice versa. The simultaneous design consisted of six or 11 training days; animals received one session/day of both trace eyeblink conditioning and water maze training. Separate groups were used for consecutive and simultaneous training. Animals trained on both tasks simultaneously were significantly facilitated in their ability to acquire the trace eyeblink conditioning task; no effect of simultaneous training was seen on the water maze task. No effect was seen on acquisition for either task when using the consecutive training design. Taken together, these findings provide insight into how the hippocampus processes information when animals learn multiple hippocampus-dependent tasks.     

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.     

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.     

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.     

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.     

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.     

Cholinergic modulation of trace conditioning trained in serial compound: A developmental analysis

In four experiments the effects of serial compound conditioning on responding to a trace-conditioned CS were evaluated using a fear conditioning paradigm. The subjects were 18- and 25-day-old Sprague-Dawley rats, previously shown to exhibit little or no trace fear conditioning. Here, animals as young as 18 days of age were shown to be capable of trace conditioning between a visual CS1 and a shock US, provided the trace interval was filled with a non-target CS2 during serial conditioning trials (CS1-->CS2-->US). To explore cholinergic mechanisms involved in trace and serial conditioning, additional experiments assessed conditioned responding following pre-training administration of the muscarinic receptor antagonist scopolamine. Scopolamine produced a dose-dependent reduction in responding to the trace CS1, regardless of whether subjects were trained with standard trace (CS1-->trace interval-->US) or serial (CS1-->CS2-->US) trials. Responding to CS2 was unaffected by scopolamine. These data suggest that central cholinergic systems are functional in the young animals, but are not normally sufficiently activated by standard trace conditioning procedures. The results suggest that serial compound conditioning can promote trace conditioning in young rats, as it does in adults, perhaps by enhancing cholinergic activity during training. Implications for the late ontogenetic emergence of trace conditioning as it relates to maturation of neural pathways and their role in the potentiating effects of a gap filler are discussed.     

Fear develops to the conditioned stimulus and to the context during classical eyeblink conditioning in rats

In classical eyeblink conditioning, non-specific emotional responses to the aversive shock unconditioned stimulus (US), which are presumed to coincide with the development of fear, occur early in conditioning and precede the emergence of eyeblink responses. This twoprocess learning model was examined by concurrently measuring fear and eyeblink conditioning in the freely moving rat. Freezing served as an index of fear in animals and was measured during the inter-trial intervals in the training context and during a tone conditioned stimulus (CS) presented in a novel context. Animals that received CS-US pairings exhibited elevated levels of fear to the context and CS early in training that decreased over sessions, while eyeblink conditioned responses (CRs) developed gradually during acquisition and decreased during extinction. Random CS-US presentations produced a similar pattern of fear responses to the context and CS as paired presentations despite low eyeblink CR percentages, indicating that fear responding was decreased independent of high levels of learned eyeblink responding The results of paired training were consistent with two-process models of conditioning that postulate that early emotional responding facilitates subsequent motor learning, but measures from random control animals demonstrate that partial CS-US contingencies produce decrements in fear despite low levels of eyeblink CRs. These findings suggest, a relationship between CS-US contingency and fear levels during eyeblink conditioning, and may serve to clarify further the role that fear conditioning plays in this simple paradigm.     

Medial auditory thalamic input to the lateral pontine nuclei is necessary for auditory eyeblink conditioning

Auditory and visual conditioned stimulus (CS) pathways for eyeblink conditioning were investigated with reversible inactivation of the medial (MPN) or lateral (LPN) pontine nuclei. In Experiment 1, Long-Evans rats were given three phases of eyeblink conditioning. Phase 1 consisted of three training sessions with electrical stimulation of the medial auditory thalamic nuclei (MATN) paired with a periorbital shock unconditioned stimulus (US). An additional session was given with a muscimol (0.5 μL, 10 mM) or saline infusion targeting the LPN followed by a recovery session with no infusions. The same training and testing sequence was then repeated with either a tone or light CS in phases 2 and 3 (counterbalanced). Experiment 2 consisted of the same training as Experiment 1 except that muscimol or saline was infused in the MPN during the retention tests. Muscimol infusions targeting the LPN severely impaired retention of eyeblink conditioned responses (CRs) to the MATN stimulation and tone CSs but only partially reduced CR percentage to the light CS. Muscimol infusions that targeted the MPN had a larger effect on CR retention to the light CS relative to MATN stimulation or tone CSs. The results provide evidence that the auditory CS pathway necessary for delay eyeblink conditioning includes the MATN-LPN projection and the visual CS pathway includes the MPN.