Bernard G. Schreurs

Trace amounts of copper in water induce β-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease

Despite the crucial role played by cholesterol and copper in nutrition and normal brain function, recent evidence indicates that they may both be important factors in the etiology of Alzheimers disease (AD). Here we provide critical evidence for the role of cholesterol and copper in AD by showing that the addition of trace amounts of copper (0.12 ppm) to water given to cholesterol-fed rabbits can induce β-amyloid (Aβ) accumulation, including senile plaque-like structures in the hippocampus and temporal lobe, and can significantly retard the ability of rabbits to learn a difficult trace conditioning task. The Aβ deposits do not affect the ability of rabbits to detect or respond to the training stimuli nor to learn a simpler delay conditioning task. Trace amounts of copper in drinking water may influence clearance of Aβ from the brain at the level of the interface between the blood and cerebrovasculature and combined with high cholesterol may be a key component to the accumulation of Aβ in the brain, having a significant impact on learning and memory. Cholesterol-fed rabbits have at least 12 pathological markers seen in AD, suggesting that the cholesterolfed rabbit is a good animal model for studying AD.

Gene expression profiles during long-term memory consolidation

Changes in gene expression have been postulated to occur during long‐term memory (LTM). We used high‐density cDNA microarrays to assess changes in gene expression 24 h after rabbit eye blink conditioning. Paired animals were presented with a 400 ms, 1000 Hz, 82 dB tone conditioned stimulus that coterminated with a 100 ms, 60 Hz, 2 mA electrical pulse unconditioned stimulus. Unpaired animals received the same conditioned and unconditioned stimuli but presented in an explicitly unpaired manner. Differences in expression levels between paired and unpaired animals in the hippocampus and cerebellar lobule HVI, two regions activated during eye blink conditioning, indicated the involvement of novel genes as well as the participation of previously implicated genes. Patterns of gene expression were validated by in situ hybridization. Surprisingly, the data suggest that an underlying mechanism of LTM involves widespread decreased, rather than increased, gene expression. These results demonstrate the feasibility and utility of a cDNA microarray system as a tool for dissecting the molecular mechanisms of associative memory.

The Effects of Cholesterol on Learning and Memory

Cholesterol is vital to normal brain function including learning and memory but that involvement is as complex as the synthesis, metabolism and excretion of cholesterol itself. Dietary cholesterol influences learning tasks from water maze to fear conditioning even though cholesterol does not cross the blood brain barrier. Excess cholesterol has many consequences including peripheral pathology that can signal brain via cholesterol metabolites, pro-inflammatory mediators and antioxidant processes. Manipulations of cholesterol within the central nervous system through genetic, pharmacological, or metabolic means circumvent the blood brain barrier and affect learning and memory but often in animals already otherwise compromised. The human literature is no less complex. Cholesterol reduction using statins improves memory in some cases but not others. There is also controversy over statin use to alleviate memory problems in Alzheimers disease. Correlations of cholesterol and cognitive function are mixed and association studies find some genetic polymorphisms are related to cognitive function but others are not. In sum, the field is in flux with a number of seemingly contradictory results and many complexities. Nevertheless, understanding cholesterol effects on learning and memory is too important to ignore.

Conditioning-specific modification of the rabbit's unconditioned nictitating membrane response

Robust classical conditioning modifies responding to the unconditioned stimulus (US) in the absence of the conditioned stimulus (CS), a phenomenon the researchers called conditioning-specific reflex modification. Unconditioned responses (URs) to periorbital stimulation varying in intensity and duration were assessed before and after 1, 3, or 6 days of paired, explicitly unpaired, or no presentations of tone and electrical stimulation. After 3 days of pairings, conditioned responding (CRs) reached 94%, and there was an increase in latency to the peak of URs. The peak latency increase was replicated in a second experiment where rabbits reached asymptotic conditioning during 6 days of pairings. There was also a conditioning-specific increase in the amplitude of URs. There were no UR changes as a function of low level of CRs following 1 day of pairings. Data suggest that there are learning-specific changes in pathways mediating the US/UR, as well as in those mediating the CS/CR.

Single-cue delay and trace classical conditioning in schizophrenia

BACKGROUND Classical conditioning provides a means of addressing mechanisms of learning and can therefore help understand the pathophysiology of memory alteration in schizophrenia. METHODS Single cue delay and trace eyeblink conditioning were used in patients with schizophrenia and matched normal control subjects to explore, respectively, cerebellar and hippocampal integrity during learning. We measured percent of conditioned (CRs) and unconditioned responses (URs), their amplitude, and onset and peak latencies. We also accounted for spontaneous blink rates and stimulus-induced responses before learning. RESULTS During delay conditioning, patients showed CRs with longer onset and peak latencies and improved efficiency compared to normal volunteers without there being differences between patients and normal control subjects in the percentage of CRs. During trace conditioning, neither group showed an increase in CRs as a function of conditioned stimulus-unconditioned stimulus pairings, in part because the level of spontaneous blink rates exceeded the level of CRs; however, patients with schizophrenia showed increased responding 150-400 msec after the conditioned stimulus and in the last 100-150 msec before the unconditioned stimulus, whereas normal control subjects showed only the latter type of responses. The former type of response was more frequent in patients with schizophrenia even before either trace or delay conditioning. CONCLUSIONS These results suggest integrity of cerebellar mechanisms underlying conditioning, although the altered timing of CRs in patients may indicate differences in the modulation of such responses. Both the greater CR onset latency during delay and the presence of early nonadaptive responses during trace are compatible with the pattern of responding seen in animals with hippocampal damage.

Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+.

LONG-TERM depression was established in cerebellar Purkinje cells using 20 pairings of a brief, high frequency train of parallel fiber stimulation with a subsequent 100 ms depolrizing current injection. Effects were assessed on the peak amplitude of Purkinje cell excitatory post synaptic potentials (EPSPs) elicited by single parallel fiber test pulses. Intradendritically recorded Purkinje cell EPSPs exhibited long-term (> 20 min) reduction in peak amplitude following paired stimulation but not if pretreated with the protein kinase C inhibitor calphostin C or iontophoretically injected with the calcium chelator EGTA. The similarity of the stimulation protocol and behavioral conditioning paradigms suggests pairing-specific long-term synaptic depression is a valuable model for understanding the cellular mechanisms underlying cerebellar cortical contributions to learning.

Conditioning-specific reflex modification of the rabbit (Oryctolagus cuniculus) nictitating membrane response: US intensity effects

Conditioning-specific reflex modification (CRM) of the rabbit’s nictitating membrane response (NMR) describes changes in responding to an unconditioned stimulus (US) when the rabbit is tested in the absence of the conditioned stimulus. Specifically, after at least 3 days of tone-electrical stimulation pairings, responses to the US increase in size, especially at intensities weaker than the training intensity. CRM is similar to classical conditioning in that it is a function of the strength of conditioning, it can be extinguished, and it can be generalized from one stimulus to another. To compare CRM and classical conditioning further, we conducted three experiments to examine the effects of US intensity (1.0, 2.0, and 4.0 mA) on CRM. CRM was weak following conditioning with 1.0 mA and extremely strong following conditioning with 2.0 mA and 4.0 mA. The data suggest that CRM is a function of US intensity and have implications for posttraumatic stress disorder, a disorder potentially modeled by CRM.

Cholesterol modifies classical conditioning of the rabbit (Oryctolagus cuniculus) nictitating membrane response.

Cholesterol plays an important role in synapse formation, receptor function, and synaptic plasticity, and animal studies show that modifying cholesterol may improve learning and memory. Other data show that feeding animals cholesterol can induce beta amyloid accumulation. Rabbits (Oryctolagus cuniculus) fed 2% cholesterol for 8 weeks were given trace conditioning of the nictitating membrane response using a 100-ms tone, a 700-ms trace, and periorbital electrical stimulation or airpuff. Rabbits fed cholesterol showed significant facilitation of trace conditioning to airpuff and conditioning-specific reflex modification to periorbital electrical stimulation and airpuff. The cholesterol-fed rabbits had beta amyloid accumulation in the cortex, but little in the hippocampus. The data suggest cholesterol had facilitative effects that outweighed potential amnesic effects of cortical beta amyloid.

Conditioning-specific reflex modification of the rabbit's nictitating membrane response and heart rate: behavioral rules, neural substrates, and potential applications to posttraumatic stress disorder.

Interest in classical conditioning is usually focused on anticipatory responses to a stimulus associated with a significant event, and it is assumed that responses to the event itself are reflexive, involuntary, and relatively invariant. However, there is compelling evidence that both the rabbit nictitating membrane response (NMR) and heart rate response (HR), well-known reflexive reactions to aversive events, can change quite dramatically as a function of learning when measured in the absence of the conditioned stimulus. In the case of NMR conditioning, a simple blink is transformed into a larger and more complex response. For HR conditioning, reflexive heart rate acceleration can actually change to heart rate deceleration. In both cases, the reflex comes to resemble the conditioned response and follows some of the same behavioral laws. This change in response to the aversive event itself or weaker forms of that event is called conditioning-specific reflex modification (CRM). CRM may force us to reevaluate the behavioral and neural consequences of classical conditioning and may have important consequences for the treatment of conditions such as posttraumatic stress disorder.

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 Alzheimers 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.