Eyeblink tract tracing with two strains of herpes simplex virus 1.

BACKGROUND: Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. NEW METHOD: We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. RESULTS AND COMPARISON WITH EXISTING METHODS: Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. CONCLUSIONS: Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Changes in membrane properties of rat deep cerebellar nuclear projection neurons during acquisition of eyeblink conditioning.

Previous studies have shown changes in membrane properties of neurons in rat deep cerebellar nuclei (DCN) as a function of development, but due to technical difficulties in obtaining viable DCN slices from adult animals, it remains unclear whether there are learning-related alterations in the membrane properties of DCN neurons in adult rats. This study was designed to record from identified DCN cells in cerebellar slices from postnatal day 25-26 (P25-26) rats that had a relatively mature sensory nervous system and were able to acquire learning as a result of tone-shock eyeblink conditioning (EBC) and to document resulting changes in electrophysiological properties. After electromyographic electrode implantation at P21 and inoculation with a fluorescent pseudorabies virus (PRV-152) at P22-23, rats received either four sessions of paired delay EBC or unpaired stimulus presentations with a tone conditioned stimulus and a shock unconditioned stimulus or sat in the training chamber without stimulus presentations. Compared with rats given unpaired stimuli or no stimulus presentations, rats given paired EBC showed an increase in conditioned responses across sessions. Whole-cell recordings of both fluorescent and nonfluorescent DCN projection neurons showed that delay EBC induced significant changes in membrane properties of evoked DCN action potentials including a reduced after-hyperpolarization amplitude and shortened latency. Similar findings were obtained in hyperpolarization-induced rebound spikes of DCN neurons. In sum, delay EBC produced significant changes in the membrane properties of juvenile rat DCN projection neurons. These learning-specific changes in DCN excitability have not previously been reported in any species or task.

Propranolol produces short-term facilitation of extinction in a rabbit model of post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a learning-based anxiety disorder with significant public health challenges due to difficulties in treating the complex, multiple symptomology. We have developed an animal model of PTSD, based on Pavlovian eyeblink conditioning in rabbits, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). We have found previously that unpaired extinction is ideal for reducing both CRs and CRM simultaneously and shows sensitivity to systemic serotonergic and glutamatergic manipulations. The following study aimed to extend our work to examine the role of the noradrenergic system, dysregulation of which is strongly implicated as part of the neurobiology of PTSD and which may also play a role in the balance shift from fear reconsolidation to extinction during treatment. The goal of the following two studies was to examine whether the ß-adrenergic receptor antagonist propranolol combined with either a full or brief course of unpaired extinction treatment could enhance extinction of CRs and/or CRM. Results showed a within-session facilitation of propranolol on extinction of CRs, particularly during the first extinction session, and a short-term enhancement of extinction of CRM when extinction treatment was brief. However, neither benefit translated to long-term extinction retention for the majority of subjects. Findings suggest that propranolol may provide the most therapeutic benefit in situations of high arousal early in treatment, which may be more important for future patient compliance rather than long-term treatment outcomes.

Delayed unpaired extinction as a treatment for hyperarousal of the rabbit nictitating membrane response and its implications for treating PTSD.

Treatment for PTSD (Post-traumatic stress disorder) is rarely available immediately after trauma and often delayed for weeks or months after an event. In a rabbit eyeblink conditioning model of PTSD, we have previously shown that presentations of a tone conditioned stimulus (CS) and shock unconditioned stimulus (US) in an explicitly unpaired manner known as unpaired extinction is effective in reducing CS responding and US hyperarousal even if shock intensity is reduced eight-fold and elicits only minimal responding. Here we determined if delayed delivery of unpaired extinction would still be effective in extinguishing hyperarousal. Rabbits were tested for sensitivity to shock before CS-US pairings and after six days of unpaired extinction presented a day, a week or a month after CS-US pairings. Hyperarousal was extinguished a day and a week after conditioning but not after a month suggesting a significant delay in "treatment" can make hyperarousal persist. We next assessed if this persistence of hyperarousal was associative by comparing rabbits given CS-US pairings to those given explicitly unpaired CS and US presentations, measuring hyperarousal a day and a month later, followed by unpaired extinction and hyperarousal assessment. After four weeks, there was an increase in responding for all rabbits but only rabbits receiving CS-US pairings showed a significant increase in associatively-mediated hyperarousal. Importantly, both paired and unpaired groups showed increased levels of responding after unpaired extinction suggesting treatment delayed for too long may no longer be effective and could cause generalized hyperarousal.

Grouping subjects based on conditioning criteria reveals differences in acquisition rates and in strength of conditioning-specific reflex modification.

Averaging behavioral data such as the nictitating membrane response (NMR) across subjects can conceal important individual and group differences. Analyses were conducted of NMR data from rabbits that were grouped based on the point during NMR conditioning when subjects produced 8 conditioned responses (CR) in a set of 10 trials. This resulted in five groups (Early Day 1, Late Day 1, Early Day 2, Late Day 2, Early Day 3) in which group differences in CR acquisition rates were found. Percent (%) CRs were not found to increase monotonically and between-session differences in % CR were found. Conditioning-specific reflex modification (CRM) of the NMR is a type of enhanced reflexive responding of the NMR that is detected when the unconditioned stimulus (US) is presented in the absence of the conditioned stimulus (CS) following paired classical conditioning. CRM occurred in some subjects in all five groups. Subjects from both the group that was fastest and the group that was slowest to reach the learning criterion had unconditioned response (UR) topographies following NMR conditioning that strongly resembled the CR-UR response sequence elicited during NMR conditioning. This finding was most pronounced when the US duration used to assess CRM was equivalent to that used during NMR conditioning, further evidence to support the hypothesis that CRM is a CR that has generalized from the CS to the US. While grouping data based on conditioning criteria did not facilitate identifying individuals more predisposed to exhibiting CRM, strong CRM only occurred in the groups that reached the conditioning criterion the fastest.

Effects of systemic glutamatergic manipulations on conditioned eyeblink responses and hyperarousal in a rabbit model of post-traumatic stress disorder.

Glutamatergic dysfunction is implicated in many neuropsychiatric conditions, including post-traumatic stress disorder (PTSD). Glutamate antagonists have shown some utility in treating PTSD symptoms, whereas glutamate agonists may facilitate cognitive behavioral therapy outcomes. We have developed an animal model of PTSD, based on conditioning of the rabbit's eyeblink response, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). The optimal treatment to reduce both CRs and CRM is unpaired extinction. The goals of the study were to examine whether treatment with the N-methyl-D-aspartate glutamate receptor antagonist ketamine could reduce CRs and CRM, and whether the N-methyl-D-aspartate agonist D-cycloserine combined with unpaired extinction treatment could enhance the extinction of both. Administration of a single dose of subanesthetic ketamine had no significant immediate or delayed effect on CRs or CRM. Combining D-cycloserine with a single day of unpaired extinction facilitated extinction of CRs in the short term while having no impact on CRM. These results caution that treatments may improve one aspect of the PTSD symptomology while having no significant effects on other symptoms, stressing the importance of a multiple-treatment approach to PTSD and of animal models that address multiple symptoms.

Effects of extinction treatments on the reduction of conditioned responding and conditioned hyperarousal in a rabbit model of posttraumatic stress disorder (PTSD).

We have previously characterized a model of posttraumatic stress disorder (PTSD), based on classical conditioning of the rabbit nictitating membrane response (NMR), that focuses on 2 key PTSD-like features: conditioned responses to trauma-associated cues and hyperarousal. In addition to the development of conditioned NMRs (CRs) to a tone conditioned stimulus (CS) associated with a periorbital shock unconditioned stimulus (US), we have observed that rabbits also exhibit a conditioning-specific reflex modification (CRM) of the NMR that manifests as an exaggerated and more complex reflexive NMR to presentations of the US by itself, particularly to intensities that elicited little response prior to conditioning. Previous work has demonstrated that unpaired presentations of the CS and US are successful at extinguishing CRs and CRM simultaneously, even when a significantly weakened version of the US is utilized. In the current study, additional extinction treatments were tested, including continued pairings of the CS with a weakened US and exposure to the training context alone, and these treatments were contrasted with the effects of unpaired extinction with a weakened US and remaining in home cages with no further treatment. Results showed that continued pairings only slightly decreased CRs and CRM, while context exposure had no effect on CRs and marginal effects on reducing CRM. Unpaired extinction was still the most effective treatment for reducing both. Findings are discussed in terms of applications to cognitive-behavioral therapies for treatment of PTSD, such as incorporating mild, innately stressful stimuli into virtual reality therapy.

Dietary cholesterol degrades rabbit long term memory for discrimination learning but facilitates acquisition of discrimination reversal.

We have shown previously that feeding dietary cholesterol before learning can improve acquisition whereas feeding cholesterol after learning can degrade long term memory. To examine these different findings within a single paradigm, we fed groups of rabbits 2% cholesterol or normal chow with or without 0.12 ppm copper added to the drinking water following two-tone discrimination learning of the nictitating membrane response in which a 8-kHz tone (conditioned stimulus, CS+) was followed by air puff and a 1-kHz tone (CS-) was not. After eight weeks on the diet, we assessed the rabbits' conditioned responding during testing and retraining. We then reversed the two-tone discrimination and assessed responding to the 1-kHz tone CS+ and the 8-kHz CS-. During testing, rabbits given cholesterol without copper had lower levels of responding to CS+ than rabbits in the other groups suggesting they did not retain the discrimination as well. However, during a brief discrimination retraining session, their response levels to the CS+ returned to the level of the other groups, demonstrating a return of the memory of the original discrimination. At the end of discrimination reversal, these same rabbits exhibited superior discrimination indexed by lower response levels to CS- but similar levels to CS+, suggesting they were better able to acquire the new relationship between the two tones by inhibiting CS- responses. These results add to our previous data by showing cholesterol diet-induced degradation of an old memory and facilitation of a new memory can both be demonstrated within a discrimination reversal paradigm. Given discrimination reversal is a hippocampally-dependent form of learning, the data support the role of cholesterol in modifying hippocampal function as we have shown previously with in vitro brain slice recordings.

Subacute fluoxetine enhances conditioned responding and conditioning-specific reflex modification of the rabbit nictitating membrane response: implications for drug treatment with selective serotonin reuptake inhibitors.

Extensive research on the rabbit nictitating membrane response (NMR) has shown that the NMR reflex can become exaggerated following classical fear conditioning. This learning-related change is referred to as conditioning-specific reflex modification (CRM) and is observed in the absence of the conditioned stimulus. The aim of the current study was to examine the sensitivity of the CRM paradigm to serotonergic manipulation with fluoxetine, a commonly prescribed selective serotonin reuptake inhibitor for anxiety disorders. To assess the effect of fluoxetine on exaggerated reflexive responding indicative of CRM and on conditioned cued fear, rabbits underwent delay NMR conditioning (pairings of tone and periorbital shock) and were tested for CRM, followed by 5 days of daily fluoxetine (0.03, 0.3, or 3.0 mg/kg) or saline injections. CRM was reassessed 1 day and 1 week later, followed by a retention test of conditioned responses (CRs) to the tone. Fluoxetine (3.0 mg/kg) enhanced CRM and retention of conditioned responses, a week after treatment ceased, and this is in agreement with the reports on increased anxiety-like behaviors in other animal models and humans. The CRM paradigm, therefore, may provide important insight into the mechanisms underlying the paradoxical selective serotonin reuptake inhibitor effects.

Ontogeny of trace eyeblink conditioning to shock-shock pairings in the rat pup.

Rats are responsive to shock from an early age, but eyeblink conditioning to a tone-conditioned stimulus (conditional stimulus; CS) paired with a shock-unconditioned stimulus (US) does not emerge until postnatal Day 20 (P20). More generalized postural responses such as conditioned freezing can occur at P16. Using the same periorbital shock as both the CS and US in a US-US conditioning paradigm previously shown to be effective in adult animals, we found that shock-shock pairings with a 200-ms trace interval resulted in eyeblink conditioning in younger animals than previously thought. Some rat pups showed conditioned eyeblink responses as early as P12, and by P18, conditioned responses were fully developed in all animals. Unpaired control subjects confirmed that responding in paired subjects was associative. Although many stimuli can act as a CS in adults, the advantage of using US-US pairings is that responses to the first US ensure young rat pups are capable of detecting the stimulus-something that may not be true when auditory or visual stimuli are used early in the development of altricial animals. The US-US pairing paradigm could be used to study the ontogeny and neural substrates of learning and memory before other sensory systems mature, and evaluate learning and memory in animal models of early developmental disorders.

Predictors of susceptibility and resilience in an animal model of posttraumatic stress disorder.

Animal models of posttraumatic stress disorder (PTSD) are based on fear conditioning where innocuous cues elicit reactions that originally occur to traumatic events--a core feature of PTSD. Another core feature is hyperarousal--exaggerated reactions to stressful events. One limitation of animal models of PTSD is that group effects do not model the sporadic incidence of PTSD. We developed an animal model of PTSD in which rabbit nictitating membrane responses become exaggerated as a function of classical conditioning to a tone conditioned stimulus (CS) paired with a shock unconditioned stimulus (US). Exaggerated responses to the US are a form of hyperarousal termed conditioning-specific reflex modification (CRM) and occur in the absence of the CS. Inspecting data across several experiments, we determined 25% of our rabbits exhibit strong CRM despite all subjects having high levels of conditioning. To determine how prone rabbits were to CRM (susceptibility) or how resistant (resilience), we examined data from 135 rabbits analyzing for factors during CS-US pairings and during US prescreening that would predict CRM. We found the magnitude of CRM was correlated with the onset latency and area of conditioned responding during CS-US pairings and with the peak latency of a response during US pretesting. In an animal model of PTSD that more accurately reflects clinical prevalence, we can begin to predict susceptibility not only during responding to a stressful conditioning situation but also during a screening process before the stressful situation takes place. The results suggest relatively innocuous testing may help detect PTSD after trauma and screen for it before trauma occurs.

Dietary Cholesterol Concentration and Duration Degrade Long-Term Memory of Classical Conditioning of the Rabbit's Nictitating Membrane Response.

A rabbit model of Alzheimer's disease based on feeding a cholesterol diet for eight weeks shows sixteen hallmarks of the disease, including learning and memory changes. Although we have shown 2% cholesterol and copper in water can retard learning, other studies show feeding dietary cholesterol before learning can improve acquisition whereas feeding cholesterol after learning can degrade long-term memory. We explored this issue by manipulating cholesterol concentration and duration following classical trace conditioning of the rabbit's nictitating membrane response and assessed conditioned responding after eight weeks on cholesterol. First, rabbits given trace classical conditioning followed by 0.5%, 1%, or 2% cholesterol for eight weeks showed body weight and serum cholesterol levels that were a function of dietary cholesterol. Although all concentrations of cholesterol showed some sign of retarding long-term memory, the level of memory retardation was correlated with serum cholesterol levels. Second, rabbits given trace conditioning followed by different durations of a 2% cholesterol diet combined with different durations of a 0% control diet for 8 weeks showed duration and timing of a 2% cholesterol diet were important in affecting recall. The data support the idea that dietary cholesterol may retard long-term memory.

Cholesterol increases ventricular volume in a rabbit model of Alzheimer's disease.

One of the hallmarks of Alzheimer's disease is a significant increase in ventricular volume. To date we and others have shown that a cholesterol-fed rabbit model of Alzheimer's disease displays as many as fourteen different pathological markers of Alzheimer's disease including amyloid-ß accumulation, thioflavin-S staining, blood brain barrier breach, microglia activation, cerebrovasculature changes, and alterations in learning and memory. Using structural magnetic resonance imaging at 3T, we now report that cholesterol-fed rabbits also show a significant increase in ventricular volume following 10 weeks on a diet of 2% cholesterol. The increase in volume is attributable in large part to increases in the size of the third ventricle. These changes are accompanied by significant increases in the number of amyloid-ß immuno-positive cells in the cortex and hippocampus. Increases in the number of amyloid-ß neurons in the cortex also occurred with the addition of 0.24 ppm copper to the drinking water. Together with a list of other pathological markers, the current results add further validity to the value of the cholesterol-fed rabbit as a non-transgenic animal model of Alzheimer's disease.

Incubation of conditioning-specific reflex modification: implications for Post Traumatic Stress Disorder.

Incubation of fear has been used to account for the delayed manifestation of symptoms of fear and anxiety including the delayed onset of Post Traumatic Stress Disorder (PTSD). We have shown the utility of classical conditioning-specific modification of the rabbit nictitating membrane response (NMR) as a model of PTSD. This modification includes an exaggeration in the size and a change in the timing of the unconditioned NMR after several days of classical conditioning. To assess the effects of incubation on conditioning-specific modification, we measured changes in responding as a function of the time between classical conditioning and NMR testing. After just one day of classical conditioning resulting in modest levels of learning, increases in response size were an inverted-U shaped function of days of incubation with little if any change occurring one and ten days after training but significant change occurring after six days. The incubation effect persisted for a week. An unpaired control group showed no change in the size of the response confirming the incubation effect was associative. The results bear a striking resemblance to symptoms of PTSD that do not always occur immediately after trauma and become exacerbated over time and then persist. They point to a window when incubation can exacerbate symptoms and speak to the vulnerability of re-experiencing trauma too soon. This could be a serious problem for military or emergency personnel recalled to combat or a disaster site without sufficient time to deal with the effects of their initial experiences.

Classical conditioning and conditioning-specific reflex modification of rabbit heart rate as a function of unconditioned stimulus location.

Heart rate conditioning is used as an index of conditioned fear and is important for understanding disorders of anxiety and stress, including post traumatic stress disorder (PTSD). One important feature of PTSD is that patients generalize conditioned fear from danger signals to safety signals especially when the two signals have overlapping features. What has not been determined is whether generalization occurs between unconditioned stimuli with overlapping features. In the current experiment, heart rate conditioning and conditioning-specific reflex modification of rabbit heart rate were examined as a function of two different unconditioned stimulus locations. Heart rate conditioning occurred at identical terminal levels whether electrical stimulation was presented near the eye or on the back. Despite different heart rate response topographies to electrical stimulation at the two locations, conditioning-specific reflex modification was detected near the eye and on the back and appeared to generalize between the locations. Interestingly, only conditioning-specific reflex modification detected on the back persisted for a week after heart rate conditioning. This persistence may be a model for some features of post traumatic stress disorder. Overgeneralization of unconditioned responses to unconditioned stimuli similar to the trauma may also be an important aspect of PTSD modeled here.

Unpaired extinction: implications for treating post-traumatic stress disorder.

Extinction of fear is important for treating stress-related conditions particularly post-traumatic stress disorder (PTSD). Although traditional extinction presents the feared stimulus by itself, there is evidence from both clinical and basic research that repeatedly presenting the feared stimulus by itself does not prevent fear from returning. This renewal or relapse can be "thwarted" by unpaired extinction-presentations of the feared stimulus and the event producing the fear. However, no matter how effective standard unpaired extinction may be in the laboratory, repeated presentation of a traumatic event is untenable. To make an unpaired extinction procedure more clinically relevant, we classically conditioned the rabbit nictitating membrane response using electrical stimulation or air puff as the unconditioned stimulus and then during unpaired extinction reduced both the intensity of the unconditioned stimulus and the days of unpaired stimulus presentations. We found unpaired extinction reduced conditioned and exaggerated unconditioned responding (an animal analog of PTSD called conditioning-specific reflex modification) and could be accomplished with a weak unconditioned stimulus as long as extended presentations were used. Surprisingly, brief presentations of a weak unconditioned stimulus or extended presentations of a strong one made the exaggerated responses stronger. One implication is that brief treatment may not just be ineffectual; it may heighten the symptoms of PTSD. Another implication is that using strong stimuli may also heighten those symptoms.

Neurovascular changes measured by time-of-flight MR angiography in cholesterol-fed rabbits with cortical amyloid beta-peptide accumulation.

PURPOSE: To test the hypothesis that narrowing of cranial blood vessels in cholesterol-fed rabbits is a function of the duration of the high cholesterol diet. Such neurovascular changes, caused by elevated serum cholesterol, are linked to stroke and Alzheimer's disease risk. MATERIALS AND METHODS: Four groups of New Zealand White rabbits were studied. Six were fed a normal diet, 19 were fed a 2% cholesterol diet with 0.12 ppm copper in the drinking water for 8 weeks, 10 weeks, or 12 weeks. Time-of-flight (TOF) MR angiography (MRA) at 3 Tesla was used to measure arterial diameters in 11 vessels. Previously published data for amyloid beta-peptide (Abeta) accumulation in the brains measured postmortem were correlated to vessel diameters. Ventricular volumes of rabbits were measured on group-averaged data. RESULTS: Several vessel diameters decreased with cholesterol diet duration. The posterior communicating arteries showed the largest significant effect. Abeta accumulation was inversely correlated with arterial diameter. Ventricular volumes between the normal diet and 12 weeks cholesterol-fed groups were not significantly different. CONCLUSION: Reduction in vessel diameter of medium-sized vessels but not large vessels was measured in these hypercholesterolemic rabbits. The vessel diameter narrowing and cortical Abeta deposition occurred before measurable ventricular enlargement.

Classical conditioning of the rabbit's nictitating membrane response is a function of the duration of dietary cholesterol.

Modifying dietary cholesterol may improve learning and memory but very high cholesterol can cause pathophysiology and death. Rabbits fed 2% cholesterol for 8, 10 or 12 weeks with 0.12 ppm copper added to distilled water and rabbits fed a normal diet without copper added to distilled water (0 weeks) were given a difficult trace classical conditioning task and an easy delay conditioning task pairing tone with corneal air puff. The majority of cholesterol-fed rabbits survived the deleterious effects of the diet but survival was an inverse function of the diet duration. Compared to controls, the level of classical conditioning and conditioning-specific reflex modification were an inverted "U"-shaped function of diet duration. Highest levels of responding occurred in rabbits on cholesterol for 10 weeks and trace conditioning was negatively correlated with the number of hippocampal beta-amyloid-positive neurons. Rabbits on the diet for 12 weeks responded at levels comparable to controls. The data provide support for the idea that dietary cholesterol may facilitate learning and memory but there is an eventual trade off with pathophysiological consequences of the diet.

High dietary cholesterol facilitates classical conditioning of the rabbit's nictitating membrane response.

Studies have shown that modifying dietary cholesterol may improve learning and that serum cholesterol levels can be positively correlated with cognitive performance. Rabbits fed a 0, 0.5, 1 or 2% cholesterol diet for eight weeks and 0.12 ppm copper added to their drinking water received trace and then delay classical conditioning pairing tone with corneal air puff during which movement of the nictitating membrane (NM) across the eye was monitored. We found that the level of classical conditioning and conditioning-specific reflex modification (CRM) as well as the number of beta amyloid-labeled neurons in the cortex and hippocampus were a function of the concentration of cholesterol in the diet. The data provide support for the idea that dietary cholesterol may facilitate learning and memory.

Cholesterol enhances classical conditioning of the rabbit heart rate response.

The cholesterol-fed rabbit is a model of atherosclerosis and has been proposed as an animal model of Alzheimer's disease. Feeding rabbits cholesterol has been shown to increase the number of beta amyloid immunoreactive neurons in the cortex. Addition of copper to the drinking water of cholesterol-fed rabbits can increase this number still further and may lead to plaque-like structures. Classical conditioning of the nictitating membrane response in cholesterol-fed rabbits is retarded in the presence of these plaque-like structures but may be facilitated in their absence. In a factorial design, rabbits fed 2% cholesterol or a normal diet (0% cholesterol) for 8 weeks with or without copper added to the drinking water were given trace classical conditioning using a tone and periorbital electrodermal stimulation to study the effects of cholesterol and copper on classical conditioning of heart rate and the nictitating membrane response. Cholesterol-fed rabbits showed significant facilitation of heart rate conditioning and conditioning-specific modification of heart rate relative to normal diet controls. Consistent with previous research, cholesterol had minimal effects on classical conditioning of the nictitating membrane response when periorbital electrodermal stimulation was used as the unconditioned stimulus. Immunohistochemical analysis showed a significant increase in the number of beta amyloid positive neurons in the cortex, hippocampus and amygdala of the cholesterol-fed rabbits. Supplementation of drinking water with copper increased the number of beta amyloid positive neurons in the cortex of cholesterol-fed rabbits but did not produce plaque-like structures or have a significant effect on heart rate conditioning. The data provide additional support for our finding that, in the absence of plaques, dietary cholesterol may facilitate learning and memory.