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.     

Deficit in selective and divided attention associated with cholinergic basal forebrain immunotoxic lesion produced by 192-saporin; motoric/sensory deficit associated with Purkinje cell immunotoxic lesion produced by OX7-saporin

The immunotoxin 192-saporin, infused intracerebroventricularly into rats, destroys cholinergic neurons in the basal forebrain nuclei. Doses required for complete cholinergic loss also kill some Purkinje cells. The immunotoxin OX7-saporin, when infused intraventricularly into rats, destroys Purkinje cells in a pattern similar to that produced by 192-saporin, without affecting cholinergic neurons. Thus, we used OX7-saporin to distinguish behavioral effects of 192-saporin due to cerebellar damage versus those due to cholinergic cell loss. Three doses of 192-saporin (1.6, 2.6, and 3.3 micrograms/rat) were chosen along with a dose of OX7-saporin (2.0 micrograms/rat) that produced Purkinje loss equivalent to the two highest doses of 192-saporin. Groups of Fischer-344 rats were trained in the multiple choice reaction time task and retested with more complex tasks after lesioning. They were also tested in the water maze, passive avoidance, acoustic startle, and open field. The OX7-saporin group exhibited changes in many tests suggesting hypermotility and sensory deficits. The 192-saporin groups differed from the OX7-saporin group when they displayed deficits in multiple choice reaction time tasks in which novel challenges were introduced, including sessions with a noise distractor, shortened and lengthened intertrial intervals, and use of nine instead of five sources of light stimulus. The 192-saporin groups showed no impairment in the other tasks. The cholinergic basal forebrain lesion may mask some of the effects of cerebellar damage up to a threshold after which effects of Purkinje cell loss predominate when 192-saporin is administered intraventricularly.     

Purkinje cell activity in the cerebellar anterior lobe after rabbit eyeblink conditioning

The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus. Rabbits were trained in an interstimulus interval discrimination procedure in which one tone signaled a 250-msec conditioned stimulus-unconditioned stimulus (CS-US) interval and a second tone signaled a 750-msec CS-US interval. All rabbits showed conditioned responses to each CS with mean onset and peak latencies that coincided with the CS-US interval. Many anterior lobe Purkinje cells showed significant learning-related activity after eyeblink conditioning to one or both of the CSs. More Purkinje cells responded with inhibition than with excitation to CS presentation. In addition, when the firing patterns of all conditioning-related Purkinje cells were pooled, it appeared that the population showed a pattern of excitation followed by inhibition during the CS-US interval. Using cholera toxin-conjugated horseradish peroxidase, Purkinje cells in recording areas were found to project to the interpositus nucleus. These data support previous studies that have suggested a role for the anterior cerebellar cortex in eyeblink conditioning as well as models of cerebellar-mediated CR timing that postulate that Purkinje cell activity inhibits conditioned response (CR) generation during the early portion of a trial by inhibiting the deep cerebellar nuclei and permits CR generation during the later portion of a trial through disinhibition of the cerebellar nuclei.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

Blocking cholesterol synthesis impairs acquisition of the classically conditioned eyeblink response

Smith-Lemli-Opitz (SLO) syndrome is a congenital disorder characterized by severe mental retardation. Patients with SLO lack 7-dehydrocholesterol (7 dH) reductase, which catalyzes the last step of cholesterol synthesis. Administration of an agent that blocks 7 dH cholesterol reductase, BM 15.966 (BM), leads to a biochemical profile which resembles that of SLO patients, i.e., lower plasma, live and brain cholesterol levels accompanied by the appearance of the precursors 7 dH and 8 dH cholesterol. In this article we address the functional consequences of chronic BM treatment on new motor learning by assessing acquisition of the classically conditioned eyeblink response. Just-weaned rats were fed BM by gavage for four months, with half of these rats given exogenous cholesterol during the last two months of BM treatment. Acquisition of the eyeblink response was impaired in BM-treated rats. Impaired acquisition of the eyeblink response was not accompanied by alterations in responsiveness to either the conditioned or unconditioned stimulus. Exogenous cholesterol, a clinically relevant countertreatment, failed to correct for the learning impairment produced by BM treatment. Chronic treatment with a cholesterol synthesis-blocking agent impaired associative learning in just-weaned rats.     

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.     

Blocking cholesterol synthesis impairs acquisition of the classically conditioned eyeblink response.

Smith-Lemli-Opitz (SLO) syndrome is a congenital disorder characterized by severe mental retardation. Patients with SLO lack 7-dehydrocholesterol (7 dH) reductase, which catalyzes the last step of cholesterol synthesis. Administration of an agent that blocks 7 dH cholesterol reductase, BM 15.766 (BM), leads to a biochemical profile which resembles that of SLO patients, i.e., lower plasma, liver, and brain cholesterol levels accompanied by the appearance of the precursors 7 dH and 8 dH cholesterol. In this article we address the functional consequences of chronic BM treatment on new motor learning by assessing acquisition of the classically conditioned eyeblink response. Just-weaned rats were fed BM by gavage for four months, with half of these rats given exogenous cholesterol during the last two months of BM treatment. Acquisition of the eyeblink response was impaired in BM-treated rats. Impaired acquisition of the eyeblink response was not accompanied by alterations in responsiveness to either the conditioned or unconditioned stimulus. Exogenous cholesterol, a clinically relevant countertreatment, failed to correct for the learning impairment produced by BM treatment. Chronic treatment with a cholesterol synthesis-blocking agent impaired associative learning in just-weaned rats.     

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.     

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.     

Intrahippocampal scopolamine impairs both acquisition and consolidation of contextual fear conditioning

Lesions of the dorsal hippocampus have been shown to disrupt both the acquisition and the consolidation of memories associated with contextual fear (fear of the place of conditioning), but do not affect fear conditioning to discrete cues (e.g., a tone). Blockade of central muscarinic cholinergic receptor activation results in selective acquisition deficits of contextual fear conditioning, but reportedly has little effect on consolidation. Here we show for the first time that direct infusion of the muscarinic cholinergic receptor antagonist, scopolamine, into the dorsal hippocampus produces a dose-dependent deficit in both acquisition and consolidation of contextual fear conditioning, while having no impact on simple tone conditioning.     

Amygdala and hippocampal activity during acquisition and extinction of human fear conditioning

Previous functional magnetic resonance imaging (fMRI) studies have characterized brain systems involved in conditional response acquisition during Pavlovian fear conditioning. However, the functional neuroanatomy underlying the extinction of human conditional fear remains largely undetermined. The present study used fMRI to examine brain activity during acquisition and extinction of fear conditioning. During the acquisition phase, participants were either exposed to light (CS) presentations that signaled a brief electrical stimulation (paired group) or received light presentations that did not serve as a warning signal (control group). During the extinction phase, half of the paired group subjects continued to receive the same treatment, whereas the remainder received light alone. Control subjects also received light alone during the extinction phase. Changes in metabolic activity within the amygdala and hippocampus support the involvement of these regions in each of the procedural phases of fear conditioning. Hippocampal activity developed during acquisition of the fear response. Amygdala activity increased whenever experimental contingencies were altered, suggesting that this region is involved in processing changes in environmental relationships. The present data show learning-related amygdala and hippocampal activity during human Pavlovian fear conditioning and suggest that the amygdala is particularly important for forming new associations as relationships between stimuli change.