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.     

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).     

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.     

Central cannabinoid receptors modulate acquisition of eyeblink conditioning

Delay eyeblink conditioning is established by paired presentations of a conditioned stimulus (CS) such as a tone or light, and an unconditioned stimulus (US) that elicits the blink reflex. Conditioned stimulus information is projected from the basilar pontine nuclei to the cerebellar interpositus nucleus and cortex. The cerebellar cortex, particularly the molecular layer, contains a high density of cannabinoid receptors (CB1R). The CB1Rs are located on the axon terminals of parallel fibers, stellate cells, and basket cells where they inhibit neurotransmitter release. The present study examined the effects of a CB1R agonist WIN55,212-2 and antagonist SR141716A on the acquisition of delay eyeblink conditioning in rats. Rats were given subcutaneous administration of 1, 2, or 3 mg/kg of WIN55,212-2 or 1, 3, or 5 mg/kg of SR141716A before each day of acquisition training (10 sessions). Dose-dependent impairments in acquisition were found for WIN55,212-2 and SR141716A, with no effects on spontaneous or nonassociative blinking. However, the magnitude of impairment was greater for WIN55,212-2 than SR141716A. Dose-dependent impairments in conditioned blink response (CR) amplitude and timing were found with WIN55,212-2 but not with SR141716A. The findings support the hypothesis that CB1Rs in the cerebellar cortex play an important role in plasticity mechanisms underlying eyeblink conditioning.     

Classical eyeblink conditioning in adulthood: effects of age and interstimulus interval on acquisition in the trace paradigm

Effects of age and interstimulus interval (ISI) were examined in eyeblink classical conditioning with a trace paradigm. Sixty young adults were trained in Experiment 1 with 6 interstimulus intervals (ISIs): 400, 900, 1,200, 1,500, 1,800, and 2,100 ms. The purpose was to find an ISI value for humans that would result in nonoptimal acquisition. ISIs of 1,200 ms and longer were nonoptimal. Adults aged 17-81 years were trained in Experiment 2 with a nonoptimal 1,800-ms ISI in the trace paradigm. Young and middle-aged subjects demonstrated equivalent levels of acquisition, and only older subjects conditioned more poorly. Aging appears to affect conditioning in the long-ISI trace paradigm as well as in the short-ISI delay paradigm.     

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.     

Acute exposure to stress improves performance in trace eyeblink conditioning and spatial learning tasks in healthy men

The present study investigated the effects of acute stress exposure on learning performance in humans using analogs of two paradigms frequently used in animals. Healthy male participants were exposed to the cold pressor test (CPT) procedure, i.e., insertion of the dominant hand into ice water for 60 sec. Following the CPT or the control procedure, participants completed a trace eyeblink conditioning task followed by a virtual navigation Morris water task (VNMWT). Hypothalamic-pituitary-adrenocortical (HPA) axis and sympathetic autonomic system (SAS) activity were assessed by measuring salivary cortisol, heart rate, and skin conductance at selected timepoints. Results revealed positive effects of stress on performance in both tasks. The stress group showed significantly more conditioned blinks than the control group during acquisition of trace eyeblink conditioning. The stress group also performed significantly better in the VNMWT than the control group, with the former showing significantly fewer failures to locate the hidden platform in the allotted time and smaller heading errors than the latter. Regression analyses revealed positive relationships between HPA axis and SAS activity during stress and eyeblink conditioning performance. Our results directly extend findings from animal studies and suggest potential physiological mechanisms underlying stress and learning.     

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.     

Cholinergic septo-hippocampal innervation is required for trace eyeblink classical conditioning

We studied the effects of a selective lesion in rats, with 192-IgG-saporin, of the cholinergic neurons located in the medial septum/diagonal band (MSDB) complex on the acquisition of classical and instrumental conditioning paradigms. The MSDB lesion induced a marked deficit in the acquisition, but not in the retrieval, of eyeblink classical conditioning using a trace paradigm. Such a deficit was task-selective, as lesioned rats were able to acquire a fixed-interval operant conditioning as controls, and was not due to nonspecific motor alterations, because spontaneous locomotion and blink reflexes were not disturbed by the MSDB lesion. The deficit in the acquisition of a trace eyeblink classical conditioning was reverted by the systemic administration of carbachol, a nonselective cholinergic muscarinic agonist, but not by lobeline, a nicotinic agonist. These results suggest a key role of muscarinic denervation on the acquisition of new motor abilities using trace classical conditioning procedures. It might also be suggested that muscarinic agents would be useful for the amelioration of some associative learning deficits observed at early stages in patients with Alzheimer's disease.     

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.     

Medial auditory thalamus inactivation prevents acquisition and retention of eyeblink conditioning

The auditory conditioned stimulus (CS) pathway that is necessary for delay eyeblink conditioning was investigated using reversible inactivation of the medial auditory thalamic nuclei (MATN) consisting of the medial division of the medial geniculate (MGm), suprageniculate (SG), and posterior intralaminar nucleus (PIN). Rats were given saline or muscimol infusions into the MATN contralateral to the trained eye before each of four conditioning sessions with an auditory CS. Rats were then given four additional sessions without infusions to assess savings from the initial training. All rats were then given a retention test with a muscimol infusion followed by a recovery session. Muscimol infusions through cannula placements within 0.5 mm of the MGm prevented acquisition of eyeblink conditioned responses (CRs) and also blocked CR retention. Cannula placements more than 0.5 mm from the MATN did not completely block CR acquisition and had a partial effect on CR retention. The primary and secondary effects of MATN inactivation were examined with 2-deoxy-glucose (2-DG) autoradiography. Differences in 2-DG uptake in the auditory thalamus were consistent with the cannula placements and behavioral results. Differences in 2-DG uptake were found between groups in the ipsilateral auditory cortex, basilar pontine nuclei, and inferior colliculus. Results from this experiment indicate that the MATN contralateral to the trained eye and its projection to the pontine nuclei are necessary for acquisition and retention of eyeblink CRs to an auditory CS.     

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.     

Galantamine facilitates acquisition of a trace-conditioned eyeblink response in healthy, young rabbits

Previous work has demonstrated that drugs increasing brain concentrations of acetylcholine can enhance cognition in aging and brain-damaged organisms. The present study assessed whether galantamine (GAL), an allosteric modulator of nicotinic cholinergic receptors and weak acetylcholinesterase inhibitor, could improve acquisition and retention of an eyeblink (EB) classical conditioning task in healthy, young animals. We trained 24 rabbits (n = 8/group) in a 1000-msec trace Pavlovian EB conditioning paradigm in which a tone conditioned stimulus (CS) was presented for 500 msec, followed by a 500-msec trace period in which no stimuli were presented. A 100-msec corneal airpuff was the unconditioned stimulus (US). Acquisition sessions, consisting of 100 trials each, occurred daily for 10 consecutive days, followed by 3 d of extinction training. Animals were treated with one of three doses of GAL (0.0-3.0 mg/kg) prior to each session. Animals that received 3.0 mg/kg GAL showed significantly more EB conditioned responses (CRs) in fewer training trials than animals receiving either 1.5 mg/kg GAL or vehicle injections. GAL had no effect on CR performance during extinction. Pseudoconditioning control experiments, consisting of 200 explicitly unpaired tone-puff presentations indicated that GAL did not increase reactivity to the CS or US. These findings indicate that GAL may improve acquisition of moderately difficult associative learning tasks in healthy young organisms.     

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.     

Children with Specific Language Impairment are not impaired in the acquisition and retention of Pavlovian delay and trace conditioning of the eyeblink response

Three converging lines of evidence have suggested that cerebellar abnormality is implicated in developmental language and literacy problems. First, some brain imaging studies have linked abnormalities in cerebellar grey matter to dyslexia and specific language impairment (SLI). Second, theoretical accounts of both dyslexia and SLI have postulated impairments of procedural learning and automatisation of skills, functions that are known to be mediated by the cerebellum. Third, motor learning has been shown to be abnormal in some studies of both disorders. We assessed the integrity of face related regions of the cerebellum using Pavlovian eyeblink conditioning in 7–11 year-old children with SLI. We found no relationship between oral language skills or literacy skills with either delay or trace conditioning in the children. We conclude that this elementary form of associative learning is intact in children with impaired language or literacy development.     

Enhanced neuronal excitability in rat CA1 pyramidal neurons following trace eyeblink conditioning acquisition is not due to alterations in I M

Previous work done by our laboratory has demonstrated a reduction of the post-burst afterhyperpolarization (AHP) and accommodation following trace eyeblink conditioning in rabbit CA1 pyramidal neurons. Our laboratory has also demonstrated a reduction in the AHP in rat CA1 pyramidal neurons following spatial learning. In the current study we have extended our findings in rabbits by showing a reduction in both the AHP and accommodation in F344 X BN rat CA1 pyramidal neurons following acquisition of trace eyeblink conditioning. A component current of the AHP, I(M), was evaluated with a specific blocker of this current, and showed no apparent contribution to the learning-related increase in neuronal excitability. Rather, a reduction in an isoproterenol-sensitive component of the AHP, presumably sI(AHP), was observed to underlie the learning-specific change.     

Acute stress facilitates trace eyeblink conditioning in C57BL/6 male mice and increases the excitability of their CA1 pyramidal neurons

The effects of stress (restraint plus tail shock) on hippocampus-dependent trace eyeblink conditioning and hippocampal excitability were examined in C57BL/6 male mice. The results indicate that the stressor significantly increased the concentration of circulating corticosterone, the amount and rate of learning relative to nonstressed conditioned mice, and the excitability of CA1 hippocampal pyramidal neurons. Behaviorally, there was no effect of the stressor on control mice that received unpaired presentations of the tone and periorbital shock, i.e., neither stressed nor nonstressed control mice showed an increase in conditioned responding that was above baseline levels. Biophysically, the stressor significantly decreased the amplitude of the post-burst afterhyperpolarization (AHP) and decreased spike frequency accommodation relative to cells from nonstressed control mice. The effect was significant for mice that were stressed either 1 h or 24 h earlier. The results suggest that the stressor increases the excitability of hippocampal pyramidal neurons and that the mechanism underlying this increase may contribute to the more rapid acquisition of hippocampally dependent eyeblink conditioning.     

Purkinje cell loss by OX7-saporin impairs acquisition and extinction of eyeblink conditioning

The current study examined the effects of globally depleting Purkinje cells in the cerebellar cortex with the immunotoxin OX7-saporin on acquisition and extinction of delay eyeblink conditioning in rats. Rats were given OX7-saporin or saline 2 wk before the start of eyeblink conditioning. The rats that reached a performance criterion of two consecutive days with 80% or greater conditioned responses were given 5 d of extinction training followed by 2 d of reacquisition training. Rats that received infusions of OX7-saporin had 77.2%-97.9% Purkinje cell loss and exhibited impaired acquisition and extinction. The amount of Purkinje cell loss was correlated with the magnitude of the acquisition and extinction impairments. The highest correlations between Purkinje cell number and the rate of acquisition were in lobule HVI and the anterior lobe. The highest negative correlation between Purkinje cell number and the percentage of conditioned responses during extinction was in the anterior lobe. The results indicate that cerebellar Purkinje cells, particularly in the anterior lobe and lobule HVI, play significant roles in acquisition and extinction of eyeblink conditioning.     

Simultaneous classical conditioning of two effector systems

The experiment reported here was an attempt to develop an experimental procedure for independent classical conditioning of two different response systems, the skin conductance response (SCR) and the eyelid response. In a differential conditioning design with human subjects (N=50) two different unconditioned stimuli were used, a white noise (US₁) and an airpuff (US₂). Two conditioned stimuli (CS₁₁ and CS₁₂) were paired with US₁, and two other CSs (CS₂₁ and CS₂₂) were paired with US₂. A special trial architecture was used to accomplish measurement of both anticipatory SCR and eyelid responses during each trial. Significant eyelid differentiation as well as significant SCR (especially second interval responses) differentiation developed.